Methods for making novel antigen binding domains

ABSTRACT

The present invention relates generally to the field of making novel antigen binding domains against infectious diseases. The present invention also relates to novel CARs that utilize the novel antigen binding domains as an extracellular element. The present invention also relates to use of the novel antigen binding domains as therapeutic agents.

This application claims the benefit of provisional application Ser. No.62/337,769 filed on May 17, 2016.

BACKGROUND OF THE INVENTION

When a new infectious disease enters a population it can create apandemic in which a large number of individual in the population becomeinfected with the disease. In part, the disease rate may be high becausethe subjects in the population have not had prior exposure to the newinfectious agent. Recent pandemics include, for example, the world wide2009 H1N1 flu outbreak, the 2014 Ebola outbreak in Western Africa, andthe world wide HIV outbreak from the 1980's to present. During diseaseoutbreaks some subjects in a population may develop immunity against theinfectious disease while others do not and non-immune subjects maysuccumb to the disease.

Chimeric Antigen Receptors are human engineered receptors that maydirect a T-cell to attack a target recognized by the CAR. For example,CAR T cell therapy has been shown to be effective at inducing completeresponses against acute lymphoblastic leukemia and other B-cell-relatedmalignancies and has been shown to be effective at achieving andsustaining remissions for refractory/relapsed acute lymphoblasticleukemia (Maude et al., NEJM, 371:1507, 2014). CARs include an antigenbinding domain that is engineered into the man made receptor to targetthe CAR to an antigen of choice.

It is an object of the invention to use CAR constructs to find novelantigen binding domains for treating diseases such as cancer, infectiousdiseases, or aging-related conditions. It is also an object of theinvention to make novel CARs using these novel antigen binding domains.It is an object of the invention to make novel antigen binding proteinsthat can be used as therapeutics against a disease.

SUMMARY OF THE INVENTION

In some embodiments, the invention relates to methods for finding newantigen binding domains against antigens associated with a disease. Insome embodiments, the invention relates to methods for finding newantigen binding domains against antigens newly associated with adisease. In some embodiments, the invention relates to identifying newtarget antigens and antigen binding domain pairs. In some embodiments,the invention related to validating new antigen targets for a disease orcondition. In some embodiments, the invention is used to find antigenbinding domains that bind antigens associated with an infectiousdisease. In some embodiments, antigen binding domains are obtained froma subject who has become immune to the infectious disease. In someembodiments, the invention is used to find antigen binding domains thatbind antigens associated with a cancer. In some embodiments, theinvention is used to find self-antigen binding domains associated withautoimmune diseases. In some embodiments, antigen binding domains areobtained from a subject whose immune system has responded to an antigen.In some embodiments, the antigen binding domain is from an antibody. Insome embodiments, the antigen binding domain is from a T-cell receptor.In some embodiments, the antigen binding domain is from a receptor suchas, for example the CD94/NKG2 receptor family (e.g., NKG2A, NKG2B,NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), the 2B4 receptor, the NKp30, NKp44,NKp46, and NKp80 receptors, the Toll-like receptors (e.g., TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RP105). In someembodiments, the antigen binding domains are obtained from thelymphocytes of a subject exposed to the antigen. In some embodiments,potential antigen binding domains are engineered into a CAR construct tobe screened for activity against a target antigen.

In some embodiments, the invention relates to CAR, Smart-CAR, DE-CAR,and/or Side-CAR constructs that do not have an extracellular element (anantigen binding domain) for use in the invention. These partial CARconstructs are called CAR chassis, and can be combined with antigenbinding domains obtained from a subject to make a library of CARconstructs with potential antigen binding domains for the diseaseantigen. In some embodiments, the CAR chassis constructs comprise anucleic acid encoding a CAR (chimeric antigen receptor) chassis with anucleic acid encoding a Destabilizing Element or a nucleic acid encodinga RNA control device. In some embodiments, the invention relates to CARchassis, Smart CAR chassis, DE-CAR chassis, and/or Smart-DE-CAR chassisthat are comprised of at least two parts which associate to form a CARchassis, Smart CAR chassis, DE-CAR chassis and/or Smart-DE-CAR chassisof the invention (called Side-CAR chassis). In some embodiments, thechassis are combined with an antigen binding domain to form CAR, SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR constructs. In someembodiments, a library of antigen binding elements are combined with thechassis for a CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR anda library of CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARconstructs are made.

In some embodiments, novel antigen binding domains are obtained fromsubjects who have become immune to an infectious agent. In someembodiments, novel antigen binding domains are obtained from subjectswho have become immune to a cancer, infectious disease, or otherimmunologic challenge. In some embodiments, novel antigen bindingdomains are obtained from subjects who have been challenged with anantigen. In some embodiments, novel antigen binding domains are obtainedfrom subjects who have had an immune response to a cancer, infectiousdisease, or other immunologic challenge. In some embodiments, immunecells from the subject are obtained and nucleic acids encoding antigenbinding proteins are obtained from the immune cells. In someembodiments, nucleic acids encoding the antigen binding proteins areantibodies and are obtained from, for example, plasma cells and memoryB-cells. In some embodiments, the nucleic acids encoding the antigenbinding proteins are T-cell receptors from, for example, cytotoxicT-cells, helper T-cells, and memory T-cells.

In some embodiments, the nucleic acids obtained from a subject are usedto make a library of nucleic acids encoding antigen binding domains. Insome embodiments, the nucleic acids encode antibody heavy and lightchains from a subject or other source (including, for example, syntheticsources). In some embodiments, the nucleic acids represent the immuneantibody repertoire of a subject who has become immune to an infectiousdisease, cancer, or other immunogenic challenge. In some embodiments,the nucleic acids represent the antibody repertoire of a subject who hashad an immune response to an infectious disease, cancer, or otherimmunogenic challenge. In some embodiments, the antibody repertoire isfrom a subject that is naïve for the target antigen. In someembodiments, the antibody repertoire represents the germ line repertoireof a subject or species. In some embodiments, the nucleic acids encodingthe heavy and light chains of the antibody are combined in acombinatorial fashion to generate many different combinations of lightchains and heavy chain. In some embodiments, the nucleic acids representthe T-cell receptor repertoire of a subject who has become immune to aninfectious disease, cancer, or other immunogenic challenge. In someembodiments, the nucleic acids represent the T-cell receptor repertoireof a subject who has had an immune reaction to an infectious disease,cancer, or other immunogenic challenge. In some embodiments, the T-cellreceptor repertoire is from a subject that is naïve for the targetantigen. In some embodiments, the T-cell receptor repertoire representsthe germ line repertoire of a subject or species. In some embodiments,the nucleic acids encoding the alpha, beta, gamma and zeta chains of theT-cell receptor are combined in appropriate combinatorial fashion togenerate a repertoire of antigen binding domains from the T-cellreceptor chains.

In some embodiments, the nucleic acids encoding the antigen bindingdomains are engineered into single chain molecules, and these nucleicacids are operably linked to CAR chassis, Smart CAR chassis, DE-CARchassis, Smart-DE-CAR chassis, and/or Side CAR chassis of the invention.In some embodiments, antigen binding domains that bind to targetantigens can be screened or selected for from the CAR, Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR constructs with the candidateantigen binding domains. In some embodiments, the CAR, Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR constructs that bind to targetantigens can be used in therapeutic applications.

In some embodiments, the target antigen of the new antigen bindingdomains is validated. In some embodiments, the antigen binding domain isused to identify and validate the target antigen. In some embodiments,the target antigen is associated with a cancer cell. In someembodiments, the target antigen is associated with an infectiousdisease. In some embodiments, the antigen binding domain is used topurify the target antigen by immunoaffinity. In some embodiments, a CAR,Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR construct with theantigen binding domain is used to identify the target antigen. In someembodiments, cancer cells, cells infected with an infectious agent,and/or infectious agents are disrupted and the disrupted materials aresubjected to purification techniques. In some embodiments, thepurification technique uses the antigen binding domain. In someembodiments, the target antigen is isolated by immune precipitation withthe antigen binding domain. In some embodiments, a column with theantigen binding domain is used to affinity purify the target antigen. Insome embodiments, purification fractions are tested using the antigenbinding domains to identify fractions with target antigen.

In some embodiments, the Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide-CAR nucleic acids of the invention are placed into an expressionvector suitable for expression in a eukaryotic cell. In someembodiments, the nucleic acid is a DNA or RNA. In some embodiments, theRNA control device, DE, and/or Side CAR are used to control expressionof the CAR in the eukaryotic cell. In some embodiments, the inducibleexpression of the CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARis used as a control in the screening or selection for antigen bindingdomains that bind to target antigen. In some embodiments, induciblecontrol is used to verify that the growth and/or reporter signaldetected is due to the particular CAR, Smart CAR, DE-CAR, Smart-DE-CAR,and/or Side CAR construct used.

In some embodiments, the eukaryotic cell comprises an expression vectorwith nucleic acids encoding Smart CAR(s), DE-CAR(s), Smart-DE-CAR(s),and/or Side-CARs of the invention. In some embodiments, the eukaryoticcell of the invention is a mammalian cell. In some embodiments, theeukaryotic cell is a human cell or a murine cell. In some embodiments,the eukaryotic cell is a cell within the hematopoietic lineage. In someembodiments, the eukaryotic cell is a T-lymphocyte, a natural killercell, a B-lymphocyte, or a macrophage. In some embodiments, theeukaryotic cell of the invention has a desired amount of CAR, DE-CAR,and/or Side-CAR polypeptide(s). In some embodiments, the eukaryotic cellhas a desired amount of CAR, DE-CAR, and/or Side-CAR polypeptide(s) onits surface. In some embodiments, the eukaryotic cell with the CAR,DE-CAR, and/or Side-CAR polypeptide(s) of the invention has a desiredamount potential of proliferative activity.

In some embodiments, the polynucleotide encoding the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side-CAR is/are integrated into a chromosome of theeukaryotic cell. In some embodiments, the polynucleotide encoding theSmart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR is present in theeukaryotic cell extrachromosomally. In some embodiments, thepolynucleotide encoding the Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide-CAR is integrated using a genome editing enzyme (CRISPR, TALEN,Zinc-Finger nuclease), and appropriate nucleic acids (including nucleicacids encoding the Smart CAR, DE-CAR, the Smart-DE-CAR, and/or SideCAR). In an embodiment, the genome editing enzymes and nucleic acidsintegrate the nucleic acid encoding the Smart CAR, DE-CAR, Smart-DE-CAR,and/or Side-CAR at a genomic safe harbor site, such as, for example, theCCR5, AAVS1, human ROSA26, or PSIP1 loci. In some embodiments, theeukaryotic cell is a human T-lymphocyte and the nucleic acid encodingthe Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR is integrated atthe CCR5 or PSIP1 loci.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram of a Smart CAR chassis.

FIG. 2 provides a schematic diagram of a DE-CAR chassis.

FIG. 3 provides a schematic diagram of a Smart-DE-CAR chassis.

DETAILED DESCRIPTION OF THE INVENTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present teachings will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimscan be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.Numerical limitations given with respect to concentrations or levels ofa substance are intended to be approximate, unless the context clearlydictates otherwise. Thus, where a concentration is indicated to be (forexample) 10 it is intended that the concentration be understood to be atleast approximately or about 10 μg.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, an “actuator element” is defined to be a domain thatencodes the system control function of the RNA control device. In someembodiments, the actuator domain encodes the gene-regulatory function.

As used herein, an “antibody” is defined to be a protein functionallydefined as a ligand-binding protein and structurally defined ascomprising an amino acid sequence that is recognized by one of skill asbeing derived from the variable region of an immunoglobulin. An antibodycan consist of one or more polypeptides substantially encoded byimmunoglobulin genes, fragments of immunoglobulin genes, hybridimmunoglobulin genes (made by combining the genetic information fromdifferent animals), or synthetic immunoglobulin genes. The recognized,native, immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes and multiple D-segments andJ-segments. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. Antibodies exist as intact immunoglobulins, as a number ofwell characterized fragments produced by digestion with variouspeptidases, or as a variety of fragments made by recombinant DNAtechnology. Antibodies can derive from many different species (e.g.,rabbit, sheep, camel, human, or rodent, such as mouse or rat), or can besynthetic. Antibodies can be chimeric, humanized, or humaneered.Antibodies can be monoclonal or polyclonal, multiple or single chained,fragments or intact immunoglobulins.

As used herein, an “antibody fragment” is defined to be at least oneportion of an intact antibody, or recombinant variants thereof, andrefers to the antigen binding domain, e.g., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and Fv fragments, scFv antibodyfragments, linear antibodies, single domain antibodies such as sdAb(either V_(L) or V_(H)), camelid VHH domains, and multispecificantibodies formed from antibody fragments. The term “scFv” is defined tobe a fusion protein comprising at least one antibody fragment comprisinga variable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the V_(L) and V_(H) variable regions in eitherorder, e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise V_(L)-linker-V_(H) or may compriseV_(H)-linker-V_(L).

As used herein, an “antigen” is defined to be a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including, but not limited to, virtually all proteins orpeptides, including glycosylated polypeptides, phosphorylatedpolypeptides, and other post-translation modified polypeptides includingpolypeptides modified with lipids, can serve as an antigen. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan will understand that any DNA, which comprises a nucleotidesequences or a partial nucleotide sequence encoding a protein thatelicits an immune response therefore encodes an “antigen” as that termis used herein. Furthermore, one skilled in the art will understand thatan antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be synthesized or can be derived from a biologicalsample, or can be a macromolecule besides a polypeptide. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a fluid with other biological components.

As used herein, the term “B-cell” or “B-lymphocyte” are usedinterchangeably and relate to lymphocytes that produce antibodies. Asused herein, B-cells include pro B-cells, pre B-cells, immature B-cells,activated B-cells, plasma cells, memory B-cells and other cells withinthe B-cell lineage.

As used herein, the terms “Chimeric Antigen Receptor” and the term “CAR”are used interchangeably. As used herein, a “CAR” is defined to be afusion protein comprising antigen recognition moieties andcell-activation elements.

As used herein, a “CAR T-cell” or “CAR T-lymphocyte” are usedinterchangeably, and are defined to be a T-cell containing thecapability of producing CAR polypeptide, regardless of actual expressionlevel. For example a cell that is capable of expressing a CAR is aT-cell containing nucleic acid sequences for the expression of the CARin the cell.

As used herein, a “destabilizing element” or a “DE” or a “Degron” areused interchangeably, and are defined to be a polypeptide sequence thatis inducibly resistant or susceptible to degradation in the cellularcontext by the addition or subtraction of a ligand, and which confersthis stability modulation to a co-translated polypeptide to which it isfused in cis.

As used herein, an “effective amount” or “therapeutically effectiveamount” are used interchangeably, and defined to be an amount of acompound, formulation, material, or composition, as described hereineffective to achieve a particular biological result.

As used herein, an “epitope” is defined to be the portion of an antigencapable of eliciting an immune response, or the portion of an antigenthat binds to an antibody. Epitopes can be a protein sequence orsubsequence that is recognized by an antibody.

As used herein, an “expression vector” and an “expression construct” areused interchangeably, and are both defined to be a plasmid, virus, orother nucleic acid designed for protein expression in a cell. The vectoror construct is used to introduce a gene into a host cell whereby thevector will interact with polymerases in the cell to express the proteinencoded in the vector/construct. The expression vector and/or expressionconstruct may exist in the cell extrachromosomally or integrated intothe chromosome. When integrated into the chromosome the nucleic acidscomprising the expression vector or expression construct will be anexpression vector or expression construct.

As used herein, the term “fluorescent protein” refers to a proteincapable of light emission when excited with an appropriateelectromagnetic radiation. Fluorescent proteins include proteins havingamino acid sequences that are either natural or engineered.

As used herein, a “hematopoietic cell” is defined to be a cell thatarises from a hematopoietic stem cell. This includes but is not limitedto myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes,erythrocytes, mast cells, myeloblasts, basophils, neutrophils,eosinophils, macrophages, thrombocytes, monocytes, natural killer cells,T lymphocytes, B lymphocytes and plasma cells.

As used herein, the term “luciferase” refers to a protein that uses achemical substrate to produce photons. In some embodiments, luciferaserefers to an enzyme or photoprotein, such as an oxygenase, thatcatalyzes a reaction that produces bioluminescence. Luciferases can berecombinant or naturally occurring, or a variant or mutant thereof.

As used herein, the term “reporter” or “reporter molecule” refers to amoiety capable of being detected indirectly or directly. Reportersinclude, without limitation, a chromophore, a fluorophore, a fluorescentprotein, a receptor, a hapten, an enzyme, and a radioisotope.

As used herein, the term “reporter gene” refers to a polynucleotide thatencodes a reporter molecule that can be detected, either directly orindirectly. Exemplary reporter genes encode, among others, enzymes,fluorescent proteins, bioluminescent proteins, receptors, antigenicepitopes, and transporters.

As used herein, the term “reporter probe” refers to a molecule thatcontains a detectable label and is used to detect the presence (e.g.,expression) of a reporter molecule. The detectable label on the reporterprobe can be any detectable moiety, including, without limitation, anisotope (e.g., detectable by PET, SPECT, etc), chromophore, andfluorophore. The reporter probe can be any detectable molecule orcomposition that binds to or is acted upon by the reporter to permitdetection of the reporter molecule.

As used herein, a “RNA control device” is defined to be an RNA moleculethat can adopt different structures and behaviors that correspond todifferent gene regulatory activities.

As used herein, a “single chain antibody” (scFv) is defined as animmunoglobulin molecule with function in antigen-binding activities. Anantibody in scFv (single chain fragment variable) format consists ofvariable regions of heavy (V_(H)) and light (V_(L)) chains, which arejoined together by a flexible peptide linker.

As used herein, a “T-lymphocyte” or T-cell” is defined to be ahematopoietic cell that normally develops in the thymus. T-lymphocytesor T-cells include, but are not limited to, natural killer T cells,regulatory T cells, helper T cells, cytotoxic T cells, memory T cells,gamma delta T cells and mucosal invariant T cells.

As used herein, “transfected” or “transformed” or “transduced” aredefined to be a process by which exogenous nucleic acid is transferredor introduced into a host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

Destabilizing Elements

Destabilizing elements (DE) are stability-affecting polypeptides capableof interacting with a small-molecule ligand, the presence, absence, oramount of which ligand is used to modulate the stability of theDE-polypeptide of interest. In some embodiments, the polypeptide ofinterest is an immunomodulatory polypeptide. In some embodiments, thepolypeptide of interest is a CAR. In some embodiments, binding of ligandby a DE-CAR reduces the degradation rate of the DE-CAR polypeptide inthe eukaryotic cell. In some embodiments, binding of ligand by theDE-CAR increases the degradation rate of the DE-CAR in the eukaryoticcell.

Destabilizing elements or DEs useful in the present invention aredescribed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, which is incorporated by reference in its entirety for allpurposes. For example, U.S. Ser. No. 15/070,352 describes DEs derivedfrom variants of the FKBP protein, variants of the DHFR protein, variantestrogen receptor binding domain (ERBD), and variant phototropin 1 ofAvena sativa (AsLOV2). Other examples of variant FKBP nucleic acids andpolypeptides are described in US published patent application20120178168 A1 published on Jul. 12, 2012, which is hereby incorporatedby reference in its entirety for all purposes. Other examples of variantDHFR nucleic acids and polypeptides are described in US published patentapplication 20120178168 A1 published on Jul. 12, 2012, which is herebyincorporated by reference in its entirety for all purposes. Otherexamples of variant ERBD nucleic acids, polypeptides, and ligands aredescribed in published US patent application 20140255361, which ishereby incorporated by reference in its entirety for all purposes. Otherexamples of variant AsLOV2 DEs are described in Bonger et al., ACS Chem.Biol. 2014, vol. 9, pp. 111-115, and Usherenko et al., BMC SystemsBiology 2014, vol. 8, pp. 128-143, which are incorporated by referencein their entirety for all purposes.

Other DEs can be derived from other ligand binding polypeptides byfusing in frame a nucleic acid encoding the ligand binding polypeptidewith a nucleic acid encoding a reporter. This construct is mutagenizedby well-known methods, and then mutants with increased or decreasedreporter activity in response to ligand binding are identified by aselection or screening. In some embodiments, variants obtained in afirst round of mutagenesis and selection/screening are furthermutagenized using random mutagenesis and/or creation of combinatoriallibraries of the amino acid substitutions obtained in the first round ofmutagenesis and/or substitution of other amino acids at the positionsidentified in the first round of mutagenesis. In some embodiments, thereporter polypeptide is a light emitting polypeptide such as greenfluorescent polypeptide (GFP). In some embodiments, the reporterpolypeptide can be used in a selection such as, for example, a reporterpolypeptide that provides a cell with antibiotic resistance or theability to grow in a certain nutrient environment or the ability to makea certain essential nutrient (e.g., the enzyme DHFR can be used inselection schemes with certain mammalian cell lines).

Other DEs can be derived from other ligand binding polypeptides using adegron as described above for ERBD. In some embodiments, a degron isfused to the C-terminus of the ligand binding polypeptide. In someembodiments, the degron is fused to the N-terminus of the ligand bindingpolypeptide. In some embodiments, the ligand binding polypeptide is aligand binding domain derived from the ligand binding polypeptide, or issome other truncated form of the ligand binding polypeptide that has theligand binding property. In some embodiments, a nucleic acid encodingthe ligand binding domain fused to a degron is fused in frame with anucleic acid encoding a reporter. This construct is mutagenized bywell-known methods, and then mutants with increased or decreasedreporter activity in response to ligand binding are identified by aselection or screening. In some embodiments, variants obtained in afirst round of mutagenesis and selection/screening are furthermutagenized using random mutagenesis and/or creation of combinatoriallibraries of the amino acid substitutions obtained in the first round ofmutagenesis and/or substitution of other amino acids at the positionsidentified in the first round of mutagenesis.

Other ligand binding polypeptides from which variants can be made foruse as DEs, include for example, enzymes, antibodies or antibodyfragments or antibody fragments engineered by recombinant DNA methodswith the variable domain, ligand binding receptors, or other proteins.Examples of enzymes include bromodomain-containing proteins, FKBPvariants, or prokaryotic DHFR variants. Examples of receptor elementsuseful in making DEs include: variant ERBD, or other receptors that haveligands which are nontoxic to mammals, especially humans.

In some embodiments, the ligand(s) for the DE are selected foroptimization of certain attributes for therapeutic attractiveness. Theseattributes include specificity to the target DE, affinity to the DE,bioavailability, stability, commercial availability, cost, availablerelated chemical, bio-orthogonality, or combinations thereof. In someembodiments, the ligands are permeable to the plasma membrane, or aretransported across the plasma membrane of a eukaryotic cell. In someembodiments, the ligand is orally dosable to a subject. In someembodiments, the ligand is inert (a pro-ligand) and is converted to theactive ligand by, for example, chemical means, electromagneticradiation, or metabolism by normal flora or the subject to produce theactive ligand. In some embodiments, the ligand has a serum half-lifegreater than 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24hours, 48 hours, 96 hours or more. In some embodiments, the ligand has aserum half-life less than 96 hours, 48 hours, 24 hours, 18 hours, 12hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour or less.In some embodiments the ligand has a serum half-life between 1 and 96hours, between 2 and 48 hours, between 8 and 36 hours, between 10 and 28hours, between 12 and 24 hours, between 12 and 48 hours, between 8 and48 hours or between 16 and 18 hours. In some embodiments, the ligand cancross the blood-brain barrier. In some embodiments, the ligand is smalland lipophilic. In some embodiments, the ligand cannot normally exist inhuman bodies or be introduced by normal diet. In some embodiments, theaffinity, as measured by Kd, of the ligands to the target DE is lessthan 1M, 500 mM, 100 mM, 50 mM, 20 mM, 10 mM, 5 mM, 1 mM, 500 μM, 100μM, 50 μM, 20 μM, 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 20 nM, 10nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM or less. In someembodiments, the affinity, as measured by Kd, of the ligands to thetarget DE is between 1M and 1 pM, between 1 mM and 1 nM, between 100 uMand 1 nM, between 10 uM and 1 nM, between 10 uM and 10 nM, between 10 uMand 100 nM, between 10 uM and 1 uM and between 50 uM and 5 uM, between 1uM and 500 nM. In some embodiments the ligand is a protein. In someembodiments, the ligand is a small molecule. In some embodiments, theligand is a nucleic acid.

RNA Control Devices

In some embodiments, the Ribonucleic acid (RNA) control devices of theinvention exhibit tunable regulation of gene expression, designmodularity, and target specificity. The RNA control devices of theinvention can act to rewire information flow through cellular networksand reprogram cellular behavior in response to changes in the cellularenvironment. In regulating polypeptide expression, the RNA controldevices of the invention can serve as synthetic cellular sensors tomonitor temporal and spatial fluctuations in the levels of diverse inputmolecules. RNA control devices represent powerful tools for constructingligand-controlled gene regulatory systems tailored to modulate theexpression of CAR, DE-CAR, Side-CAR and/or other polypeptides of theinvention in response to specific effector molecules enabling RNAregulation of target CAR, DE-CAR, Side-CAR and/or other polypeptideconstructs in various living systems.

The RNA control devices of the invention may be either trans-acting orcis-acting. By trans-acting, it is meant that the RNA control deviceexerts its ligand-dependent activity on a molecule, e.g. another nucleicacid, that is different from the RNA control device, e.g. not linkedthrough a phosphodiester (or equivalent) backbone linker, and even morepreferably not covalently linked to the RNA control device at all. Bycis-acting, it is meant that the RNA control device exerts itsligand-dependent activity on the same contiguous nucleic acid, i.e., anucleic acid that is covalently linked to the RNA control device, e.g.,through a phosphodiester (or equivalent) backbone linker.

In some embodiments, the RNA control devices of the invention comprise aregulatory element and a sensor element. In some embodiments, the RNAcontrol devices of the invention comprise a single element with both aregulatory and sensory function. In some embodiments, the RNA controldevices of the invention comprise a regulatory function and a sensoryfunction. In some embodiments, the RNA control devices of the inventioncomprise a regulatory element, a sensor element, and an informationtransmission element (ITE) that functionally couples the regulatoryelement and the sensor element. In some embodiments, the ITE of thesubject invention is based on, for example, a strand-displacementmechanism, an electrostatic interaction, a conformation change, or asteric effect. In some embodiments, the sensing function of the RNAcontrol device leads to a structural change in the RNA control device,leading to altered activity of the regulatory function. Some mechanismswhereby these structural changes can occur include steric effects,hydrophobicity driven effects (log p), electrostatically driven effects,nucleotide modification effects (such as methylation, etc.), secondaryligand interaction effects and other effects. In some embodiments, astrand-displacement mechanism uses competitive binding of two nucleicacid sequences (e.g., the competing strand and the RNA control devicestrand) to a general transmission region of the RNA control device(e.g., the base stem of the aptamer) to result in disruption orrestoration of the regulatory element in response to ligand binding tothe sensor element.

In some embodiments, the sensor element-regulated nucleic acids aredesigned such that it can adopt at least two distinct conformations. Inone conformation, the sensor element is capable of binding to a ligand,and the regulatory element may be in one activity state (e.g., moreactive state or less active state). In the other conformation, thesensor element is incapable of binding to the ligand, and regulatoryelement may be in another activity state. The conformation change of thesensor element may be transmitted through the information transmissionelement to the coupled regulatory element, so that the regulatoryelement adopts one of the two activity states depending on whether thesensor element can or cannot bind the ligand.

In some embodiments, the aptamer-regulated nucleic acid platform isfully modular, enabling ligand response and regulatory function (e.g.,transcript targeting) to be engineered by swapping elements within thesubject regulated nucleic acid. This provides a platform for theconstruction of tailor-made sensor element regulated nucleic acids for avariety of different ligands. Ligand binding of the sensor element insensor-regulated nucleic acids is designed separately from the targetingcapability of the regulatory element by swapping only the sensorelement. Likewise, the targeting capability of the regulatory elementcan be designed separately from the ligand binding of the sensor elementby swapping the regulatory element so that a different gene or moleculeis targeted without affecting the sensor element. Thus, the subjectsensor element-regulated nucleic acids present a powerful, flexiblemethod of tailoring spatial and temporal gene expression in both naturaland engineered contexts.

In some embodiments, the RNA control devices are cis-acting RNAsequences that regulate the production of cognate protein encoded by amessenger RNA (mRNA). In some embodiments RNA control devices compriseRNA with sequences that enable direct or indirect binding of a ligand.In some embodiments, binding of a ligand to the RNA control deviceincreases or decreases the amount of protein translated from the mRNA.In some embodiments, RNA control devices comprise riboswitches which aresegments of mRNA that bind a small molecule.

An example of an RNA control device is the theophylline responsiveswitch, comprising an aptamer (a ligand binding component) andhammerhead ribozyme (gene regulating component) (Win and Smolke 2007PNAS 104 (36): 14283-88, which is hereby incorporated by reference inits entirety for all purposes). Upon aptamer binding of theophylline,the ribozyme becomes inactive and enables the expression of the desiredtransgene. In the absence of theophylline the ribozyme self cleaves,leading to nuclease driven degradation of the mRNA, inhibitingtranslation of the mRNA into protein.

In some embodiments, the RNA control device comprises a sensor elementand a regulatory element. In some embodiments the sensor element is anRNA aptamer. In some embodiments, the RNA control device comprises morethan one sensor element. In some embodiments the regulatory element is aribozyme. In some embodiments the ribozyme is a hammerhead ribozyme. Insome embodiments, the ribozyme is a hairpin ribozyme, or a hepatitisdelta virus (HDV) ribozyme, or a Varkud Satellite (VS) ribozyme, or aglmS ribozyme. In other embodiments the ribozyme is a ribozyme known inthe art.

In some embodiments, the RNA control device is embedded within a nucleicacid that encodes a transgene. In some embodiments the transgene ofinterest encodes a chimeric antigen receptor, a DE-chimeric antigenreceptor, or a Side CAR.

In some embodiments an RNA control device or devices are embedded withina DNA sequence. In some embodiments, the RNA control device is encodedfor in messenger RNA. In some embodiments multiple RNA control devicesare encoded in cis with a transgene-encoding mRNA. In some embodiments,the RNA control device is repeated. In some embodiments the nucleic acidthat is used to encode the RNA control device is repeated. By includingmultiple RNA control devices, sensitivity and dose response may betailored or optimized. In some embodiments multiple RNA control devicesare included, with each RNA control device being specific for adifferent ligand. This embodiment can mitigate unintentional expressiondue to endogenously produced ligands that interact with the sensorelement.

RNA Control Devices: Sensor Elements

Sensor elements useful in the present invention are described in U.S.patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, which isincorporated by reference in its entirety for all purposes. In someembodiments, an “aptamer” is a nucleic acid molecule, such as RNA or DNAthat is capable of binding to a specific molecule with high affinity andspecificity (Ellington et al., Nature 346, 818-22 (1990); and Tuerk etal., Science 249, 505-10 (1990), which are hereby incorporated byreference in their entirety for all purposes). For a review of aptamersthat recognize small molecules, see Famulok, Science 9:324-9 (1999),which is hereby incorporated by reference in its entirety for allpurposes.

In some embodiments, the binding affinity of the aptamer for its ligandis sufficiently strong and the structure formed by the aptamer whenbound to its ligand is significant enough so as to switch an RNA controldevice of the invention between “on” and “off” states. In someembodiments, the association constant for the aptamer and associatedligand is preferably such that the ligand functions to bind to theaptamer and have the desired effect at the concentration of ligandobtained upon administration of the ligand to a subject. For in vivouse, for example, the association constant should be such that bindingoccurs well below the concentration of ligand that can be achieved inthe serum or other tissue, preferably well below the concentration ofligand that can be achieved intracellularly since cellular membranes maynot be sufficiently permeable to allow the intracellular ligandconcentration to approach the level in the serum or extracellularenvironment. In some embodiments, the required ligand concentration forin vivo use is also below that which could have undesired effects on theorganism.

Ligands for RNA Control Devices

RNA control devices can be controlled via the addition of exogenous orendogenous ligands. In some embodiments, the ligands are selected foroptimization of certain attributes for therapeutic attractiveness. Theseattributes include specificity to the target RNA control device,affinity to the RNA control device, bioavailability, stability,commercial availability, cost, available related chemical,bio-orthogonality, or combinations thereof. In some embodiments, theligands are permeable to the plasma membrane, or are transported acrossthe plasma membrane of a eukaryotic cell. In some embodiments, theligand is orally dosable to a subject. In some embodiments, the ligandis inert (a pro-ligand) and is metabolized by normal flora or thesubject to produce the active ligand. In some embodiments, the ligandhas a serum half-life greater than 1 hour, 2 hours, 4 hours, 6 hours, 8hours, 12 hours, 24 hours, 48 hours, 96 hours or more. In someembodiments, the ligand has a serum half-life less than 96 hours, 48hours, 24 hours, 18 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4hours, 2 hours or 1 hour or less. In some embodiments the ligand has aserum half-life between 1 and 96 hours, between 2 and 48 hours, between8 and 36 hours, between 10 and 28 hours, between 12 and 24 hours,between 12 and 48 hours, between 8 and 48 hours or between 16 and 18hours. In some embodiments, the ligand can cross the blood-brainbarrier. In some embodiments, the ligand is small and lipophilic. Insome embodiments, the ligand cannot normally exist in human bodies or beintroduced by normal diet. In some embodiments, the affinity, asmeasured by Kd, of the ligands to the target RNA control device is lessthan 1M, 500 mM, 100 mM, 50 mM, 20 mM, 10 mM, 5 mM, 1 mM, 500 μM, 100μM, 50 μM, 20 μM, 10 μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 20 nM, 10nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM or less. In someembodiments, the affinity, as measured by Kd, of the ligands to thetarget DE is between 1M and 1 pM, between 1 mM and 1 nM, between 100 uMand 1 nM, between 10 uM and 1 nM, between 10 uM and 10 nM, between 10 uMand 100 nM, between 10 uM and 1 uM and between 50 uM and 5 uM, between 1uM and 500 nM. In some embodiments the ligand is a protein. In someembodiments, the ligand is a small molecule. In some embodiments, theligand is a nucleic acid.

In some embodiments, the ligand is a naturally occurring, secretedmetabolite. For example, a ligand that is uniquely produced by a tumor,or present in the tumor microenvironment is the ligand for the sensorelement and binding of this ligand to the sensor element changes theactivity of the RNA control device. Thus the control device isresponsive and controlled through chemical signaling or proximity to atumor.

In some embodiments, the ligand is selected for its pharmacodynamic orADME behavior. For example ligands may be preferentially localized tospecific portions of the human anatomy and physiology. For examplecertain molecules are preferentially absorbed or metabolized in the gut,the liver, the kidney etc. In some embodiments the ligand is selected todemonstrate preferential pharmacodynamic behavior in a particular organ.For example, it would be useful to have a ligand that preferentiallylocalizes to the colon for a colorectal carcinoma so that the peakconcentration of the ligand is at the required site, whereas theconcentrations in the rest of the body is minimized, preventingundesired, nonspecific toxicity. In some embodiments the ligand isselected to demonstrate non preferential pharmacodynamic behavior. Forexample, for disseminated tumors like hematological malignancies, itwould be useful to have non variant concentration of the ligandthroughout the body.

RNA Control Devices: Regulatory Elements

In some embodiments, the regulatory element comprises a ribozyme, or anantisense nucleic acid, or an RNAi sequence or precursor that gives riseto a siRNA or miRNA, or a shRNA or precursor thereof, or an RNAse IIIsubstrate, or an alternative splicing element, or a transcriptionterminator, or a ribosome binding site, or an IRES, or a polyA site.Regulatory elements useful in the present invention are described inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016,which is incorporated by reference in its entirety for all purposes.

General approaches to constructing oligomers useful in antisensetechnology have been reviewed, for example, by van der Krol et al.(1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668, which are hereby incorporated by reference in theirentirety for all purposes. Certain miRNAs that may be used in theinvention are described in Brennecke et al., Genome Biology 4:228(2003); Kim et al., Mol. Cells. 19:1-15 (2005), which are herebyincorporated by reference in their entirety for all purposes.

In some embodiments, the RNA control devices have multiple regulatoryelements, and/or multiple sensor elements. In some embodiments, themultiple sensor elements recognize different ligands. In someembodiments, the multiple sensor elements have different effects on theregulatory element.

Chimeric Antigen Receptors

In some embodiments, chimeric antigen receptors (CARs) are fusedproteins comprising an extracellular antigen-binding/recognitionelement, a transmembrane element that anchors the receptor to the cellmembrane and at least one intracellular element. These CAR elements areknown in the art, for example as described in patent applicationUS20140242701, which is incorporated by reference in its entirety forall purposes herein. In some embodiments, the CAR of the invention is arecombinant polypeptide construct comprising at least an extracellularantigen binding element, a transmembrane element and an intracellularsignaling element comprising a functional signaling element derived froma stimulatory molecule. In some embodiments, the stimulatory molecule isthe zeta chain associated with the T cell receptor complex. In someembodiments, the cytoplasmic signaling element further comprises one ormore functional signaling elements derived from at least onecostimulatory molecule. In some embodiments, the costimulatory moleculeis chosen from 4-1BB (i.e., CD137), CD27 and/or CD28. In someembodiments, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition element, a transmembrane element andan intracellular signaling element comprising a functional signalingelement derived from a stimulatory molecule. In some embodiments, theCAR comprises a chimeric fusion protein comprising an extracellularantigen recognition element, a transmembrane element and anintracellular signaling element comprising a functional signalingelement derived from a co-stimulatory molecule and a functionalsignaling element derived from a stimulatory molecule. In someembodiments, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition element, a transmembrane element andan intracellular signaling element comprising two functional signalingelements derived from one or more co-stimulatory molecule(s) and afunctional signaling element derived from a stimulatory molecule. Insome embodiments, the CAR comprises a chimeric fusion protein comprisingan extracellular antigen recognition element, a transmembrane elementand an intracellular signaling element comprising at least twofunctional signaling elements derived from one or more co-stimulatorymolecule(s) and a functional signaling element derived from astimulatory molecule. In some embodiments, the CAR comprises an optionalleader sequence at the amino-terminus (N-term) of the CAR fusionprotein. In some embodiments, the CAR further comprises a leadersequence at the N-terminus of the extracellular antigen recognitionelement, wherein the leader sequence is optionally cleaved from theantigen recognition element (e.g., a scFv) during cellular processingand localization of the CAR to the cellular membrane.

Chimeric Antigen Receptor—Extracellular Element

Extracellular elements useful in the present invention are described inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016,which is incorporated by reference in its entirety for all purposes.

In some embodiments, the extracellular element(s) can be obtained fromthe repertoire of antibodies obtained from the immune cells of a subjectthat has become immune to a disease, such as for example, an infectiousdisease, cancer, or other diseases. In some embodiments, a library ofextracellular element-CARs is made from the repertoire of antibodiesobtained from the immune cells of a subject that has become immune to adisease. In some embodiments, the subject has become immune to aninfectious disease. In some embodiments, the extracellular element mayconsist of an Ig heavy chain which may in turn be covalently associatedwith Ig light chain by virtue of the presence of CH1 and hinge regions,or may become covalently associated with other Ig heavy/light chaincomplexes by virtue of the presence of hinge, CH2 and CH3 domains. Insome embodiments, the extracellular element(s) can be obtained from therepertoire of T-cell receptors obtained from the immune cells of asubject that has become immune to a disease or had an immune reaction toa disease. In some embodiments, a library of extracellular element-CARsis made from the repertoire of T-cell receptors obtained from the immunecells of a subject that has become immune to a disease or had an immunereaction to a disease.

In some embodiments, the antigen binding domain is from a receptor suchas, for example the CD94/NKG2 receptor family (e.g., NKG2A, NKG2B,NKG2C, NKG2D, NKG2E, NKG2F, NKG2H), the 2B4 receptor, the NKp30, NKp44,NKp46, and NKp80 receptors, the Toll-like receptors (e.g., TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RP105). In someembodiments, a library of extracellular element-CARs is made from innateimmunity receptors from natural killer cells and/or Toll-like receptorsfrom natural killer cells, dendritic cells, macrophages, T-cells, andB-cells that are obtained from a subject who has become immune to adisease or had an immune reaction to a disease.

As described in U.S. Pat. Nos. 5,359,046, 5,686,281 and 6,103,521 (whichare hereby incorporated by reference in their entirety for allpurposes), the extracellular element may be obtained from any of thewide variety of extracellular proteins (including receptors, membranebound ligands, and other proteins associated with the membrane) orsecreted proteins associated with ligand binding and/or signaltransduction. In some embodiments, the extracellular element is part ofa protein which is monomeric, homodimeric, heterodimeric, or associatedwith a larger number of proteins in a non-covalent complex. In someembodiments, extracellular proteins for use as extracellular elementsare molecular complexes of proteins where only one chain has the majorrole of binding to ligand. In this embodiment, the extracellular elementcan be derived from the extracellular portion of the ligand bindingprotein. In some embodiments, the extracellular protein is a complex ofextracellular portions from several proteins that may be covalentlybonded through disulfide linkages. In this embodiment, the extracellularelement of a CAR may also provide for the formation of such multimericextracellular complexes. In some embodiments, the extracellular elementis comprised of truncated portions of the extracellular protein, wheresuch truncated portion is functional for binding ligand.

In some embodiments, there is provided a Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side-CAR capable of binding to an antigen derivedfrom Retroviridae (e.g. human immunodeficiency viruses such as HIV-1 andHIV-LP), Picornaviridae (e.g. poliovirus, hepatitis A virus,enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubellavirus, coronavirus, vesicular stomatitis virus, rabies virus, ebolavirus, parainfluenza virus, mumps virus, measles virus, respiratorysyncytial virus, influenza virus, hepatitis B virus, parvovirus,Adenoviridae, Herpesviridae [e.g. type 1 and type 2 herpes simplex virus(HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus],Poxviridae (e.g. smallpox virus, vaccinia virus, and pox virus), orhepatitis C virus.

In some embodiments, antigens specific for infectious diseases targetedby the Smart CAR(s), DE-CAR(s), Smart-DE-CAR(s), and/or Side-CARs of theinvention include but are not limited to any one or more of anthraxtoxin, clumping factor A, cytomegalovirus, cytomegalovirus glycoproteinB, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis Bvirus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid,Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytialvirus and TNF-α. Other antigens specific for infectious diseases will beapparent to those of skill in the art and may be used in connection withalternate embodiments of the invention.

In some embodiments, there is provided a Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR capable of binding to an antigenassociated with a bacterial strain of Staphylococci, Streptococcus,Escherichia coli, Pseudomonas, or Salmonella. In some embodiments, aphagocytic immune cell is engineered with a Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR specific for these or other pathogenicbacteria. Such Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARengineered immune cells are useful in treating bacterial infections.Examples of bacterial pathogens that can be targeted by such SmartCAR(s), DE-CAR(s), Smart-DE-CAR(s), and/or Side-CARs include,Staphylococcus aureus, Neisseria gonorrhoeae, Streptococcus pyogenes,Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae),Streptococcus pneumoniae, and Clostridium tetani. In some embodiments,there is provided a Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARcapable of binding to an antigen found on host cells infected with aninfectious pathogen (e.g., a virus, a bacteria, a protozoan, or afungus). Examples of bacterial pathogens that may infect host cellsinclude, Helicobacter pyloris, Legionella pneumophilia, a bacterialstrain of Mycobacteria spp. (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, or M. gordonea), Neisseria meningitides,Listeria monocytogenes, R. rickettsia, Salmonella spp., Brucella spp.,Shigella spp., or certain E. coli strains or other bacteria that haveacquired genes with invasive factors. Examples of viral pathogens thatmay infect host cells include, Retroviridae (e.g. human immunodeficiencyviruses such as HIV-1 and HIV-LP), Picornaviridae (e.g. poliovirus,hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, andechovirus), rubella virus, coronavirus, vesicular stomatitis virus,rabies virus, ebola virus, parainfluenza virus, mumps virus, measlesvirus, respiratory syncytial virus, influenza virus, hepatitis B virus,parvovirus, Adenoviridae, Herpesviridae [e.g. type 1 and type 2 herpessimplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), andherpes virus], Poxviridae (e.g. smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus.

In some embodiments, there is provided a Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR capable of binding to a tumor antigen suchas any one or more of 4-1BB, 5T4, adenocarcinoma antigen,alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonicanhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD21, CD22, CD23(IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51,CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3,FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factorreceptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6,insulin-like growth factor I receptor, alpha 5β1-integrin, integrinαvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid,NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostatic carcinoma cells,RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF β2,TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1,VEGFR2, 707-AP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IXantibody, CAMEL, CAP-1, CASP-8, CD25, CDC27/m, CDK4/m, CT, Cyp-B, DAM,ErbB3, ELF2M, EMMPRIN, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE,HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IL-2R,IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R,myosin/m, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minorbcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1or SART-3, survivin, TPI/m, TRP-1, TRP-2, TRP-2/INT2, WT1, NY-Eso-1 orNY-Eso-B or vimentin. Other antigens specific for cancer will beapparent to those of skill in the art and may be used in connection withalternate embodiments of the invention.

In some embodiments, antigens specific for senescent cells are targetedby the CAR, DE-CAR, Smart-DE-CAR, and/or Side-CARs of the inventioninclude but are not limited to any one or more of DEP1, NTAL, EBP50,STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A, or PLD3. Other antigensspecific for senescent cells will be apparent to those of skill in theart and may be used in connection with alternate embodiments of theinvention. See, e.g., Althubiti et al., Cell Death and Disease vol. 5,p. e1528 (2014), which is incorporated by reference in its entirety forall purposes.

Other targets for extracellular elements are described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, which isincorporated by reference in its entirety for all purposes.

Intracellular Element

In some embodiments, the intracellular element is a molecule that cantransmit a signal into a cell when the extracellular element of theSmart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR binds to (interactswith) an antigen. In some embodiments, the intracellular signalingelement is generally responsible for activation of at least one of thenormal effector functions of the immune cell in which the Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR has been introduced. The term“effector function” refers to a specialized function of a cell. Effectorfunction of a T cell, for example, may be cytolytic activity or helperactivity including the secretion of cytokines. Thus the term“intracellular signaling element” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While the entire intracellular signalingdomain can be employed, in many cases the intracellular element orintracellular signaling element need not consist of the entire domain.To the extent that a truncated portion of the intracellular signalingdomain is used, such truncated portion may be used as long as ittransduces the effector function signal. The term intracellularsignaling element is thus also meant to include any truncated portion ofthe intracellular signaling domain sufficient to transduce the effectorfunction signal. Examples of intracellular signaling elements for use inthe Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR of the inventioninclude the cytoplasmic sequences of the T cell receptor (TCR) andco-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

Intracellular elements and combinations polypeptides useful with or asintracellular elements are described in U.S. patent application Ser. No.15/070,352 filed on Mar. 15, 2016, which is incorporated by reference inits entirety for all purposes.

Transmembrane Element and Spacer Element

The Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR of the presentinvention comprises a transmembrane element. The transmembrane elementis attached to the extracellular element of the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR. In some embodiments, a transmembraneelement includes one or more additional amino acids adjacent to thetransmembrane region, e.g., one or more amino acid associated with theextracellular region of the protein from which the transmembrane wasderived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of theextracellular region) and/or one or more additional amino acidsassociated with the intracellular region of the protein from which thetransmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 upto 15 amino acids of the intracellular region). In some embodiments, thetransmembrane element is associated with one of the other elements usedin the Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR. In someembodiments, the transmembrane element is selected or modified by aminoacid substitution to avoid binding of such elements to the transmembraneelements of the same or different surface membrane proteins, e.g., tominimize interactions with other members of the receptor complex. Insome embodiments, the transmembrane element is capable ofhomodimerization with another Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR on the cell surface. In some embodiments, the amino acidsequence of the transmembrane element may be modified or substituted soas to minimize interactions with the binding elements of the nativebinding partner present in the same cell.

The transmembrane element may be contributed by the protein contributingthe multispecific extracellular inducer clustering element, the proteincontributing the effector function signaling element, the proteincontributing the proliferation signaling portion, or by a totallydifferent protein. For the most part it will be convenient to have thetransmembrane element naturally associated with one of the elements. Insome cases it will be desirable to employ the transmembrane element ofthe ζ or FcεR1γ chains which contain a cysteine residue capable ofdisulfide bonding, so that the resulting chimeric protein will be ableto form disulfide linked dimers with itself, or with unmodified versionsof the ζ, η or FcεR1γ chains or related proteins. In some embodiments,the transmembrane element will be selected or modified by amino acidsubstitution to avoid binding of such elements to the transmembraneelements of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex. In someembodiments it will be desirable to employ the transmembrane element ofζ, η, FcεR1-γ and -β, MB1 (Igα), B29 or CD3-γ, ζ, or ε, in order toretain physical association with other members of the receptor complex.

Transmembrane elements useful in the present invention are described inU.S. patent application Ser. No. 15/070,352 filed on Mar. 15, 2016,which is incorporated by reference in its entirety for all purposes.

Chimeric Antigen Receptors Coupled with Destabilizing Elements (DE-CAR)

In some embodiments of the present invention, destabilizing elements, asdescribed above, are combined in cis with a CAR, as described above, sothat the amount of the CAR polypeptide in the eukaryotic cell is underthe control of the DE. This is one embodiment of the DE-CAR of theinvention. DE-CARs, selection of DEs, and use of one or multiple DEs inthe present invention are described in U.S. patent application Ser. No.15/070,352 filed on Mar. 15, 2016, which is incorporated by reference inits entirety for all purposes.

Chimeric Antigen Receptors: Side-CARs

In some embodiments, the CARs, Smart CARs, DE-CAR, and/or Smart-DE-CARsof the invention are comprised of at least two parts which associate toform a functional CAR or DE-CAR. In some embodiments, the extracellularantigen binding element is expressed as a separate part from thetransmembrane element, optional spacer, and the intracellular element ofa CAR. In some embodiments, the separate extracellular binding elementis associated with the host cell membrane (through a means other than atransmembrane polypeptide). In some embodiments, the intracellularelement is expressed as a separate part from the extracellular element,transmembrane element, and optionally the spacer. In some embodimentsthe extracellular element and intracellular element are expressedseparately and each has a transmembrane element, and optionally aspacer. In some embodiments, each part of the CAR or DE-CAR has anassociation element (“Side-CAR”) for bringing the two parts together toform a functional CAR or DE-CAR.

Side CARs, selection of Side CARs, and their use with or without atether are described in U.S. patent application Ser. No. 15/070,352filed on Mar. 15, 2016, which is incorporated by reference in itsentirety for all purposes.

Lymphocyte Expansion Molecule and Other Regulatory Factors

The use of DEs and/or RNA control devices in the invention to controlexpression of lymphocyte expansion molecule (“LEM”), IL1, IL2, IL4, IL5,IL6, IL7, IL10, IL12, IL15, GM-CSF, G-CSF, TNFα, and/or IFNγ isdescribed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, which is incorporated by reference in its entirety for allpurposes.

Eukaryotic Cells

In the present invention, various eukaryotic cells can be used as theeukaryotic cell of the invention. In some embodiments, the eukaryoticcells of the invention are animal cells. In some embodiments, theeukaryotic cells are mammalian cells, such as mouse, rat, rabbit,hamster, porcine, bovine, feline, or canine. In some embodiments, themammalian cells are cells of primates, including but not limited to,monkeys, chimpanzees, gorillas, and humans. In some embodiments, themammalians cells are mouse cells, as mice routinely function as a modelfor other mammals, most particularly for humans (see, e.g., Hanna, J. etal., Science 318:1920-23, 2007; Holtzman, D. M. et al., J Clin Invest.103(6):R15-R21, 1999; Warren, R. S. et al., J Clin Invest. 95:1789-1797, 1995; each publication is incorporated by reference in itsentirety for all purposes). Animal cells include, for example,fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung),keratinocytes, hepatocytes, adipocytes, endothelial cells, andhematopoietic cells. In some embodiments, the animal cells are adultcells (e.g., terminally differentiated, dividing or non-dividing) orembryonic cells (e.g., blastocyst cells, etc.) or stem cells. In someembodiments, the eukaryotic cell is a cell line derived from an animalor other source.

In some embodiments, the eukaryotic cell is a cell found in thecirculatory system of a mammal, including humans. Exemplary circulatorysystem cells include, among others, red blood cells, platelets, plasmacells, T-cells, natural killer cells, B-cells, macrophages, neutrophils,or the like, and precursor cells of the same. As a group, these cellsare defined to be circulating eukaryotic cells of the invention. In someembodiments, the eukaryotic cells are derived from any of thesecirculating eukaryotic cells. The present invention may be used with anyof these circulating cells or eukaryotic cells derived from thecirculating cells. In some embodiments, the eukaryotic cell is a T-cellor T-cell precursor or progenitor cell. In some embodiments, theeukaryotic cell is a helper T-cell, a cytotoxic T-cell, a memory T-cell,a regulatory T-cell, a natural killer T-cell, a mucosal associatedinvariant T-cell, a gamma delta T cell, or a precursor or progenitorcell to the aforementioned. In some embodiments, the eukaryotic cell isa natural killer cell, or a precursor or progenitor cell to the naturalkiller cell. In some embodiments, the eukaryotic cell is a B-cell, or aplasma cell, or a B-cell precursor or progenitor cell. In someembodiments, the eukaryotic cell is a neutrophil or a neutrophilprecursor or progenitor cell. In some embodiments, the eukaryotic cellis a megakaryocyte or a precursor or progenitor cell to themegakaryocyte. In some embodiments, the eukaryotic cell is a macrophageor a precursor or progenitor cell to a macrophage.

In some embodiments, a source of cells is obtained from a subject. Thesubject may be any living organisms. In some embodiments, the cells arederived from cells obtained from a subject. Examples of subjects includehumans, dogs, cats, mice, rats, and transgenic species thereof. In someembodiments, T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In some embodiments, any number ofT cell lines available in the art, may be used. In some embodiments, Tcells can be obtained from a unit of blood collected from a subjectusing any number of techniques known to the skilled artisan, such asFicoll separation. In some embodiments, cells from the circulating bloodof an individual are obtained by apheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In some embodiments, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In some embodiments, the cells are washed with phosphate buffered saline(PBS). In an alternative aspect, the wash solution lacks calcium and maylack magnesium or may lack many if not all divalent cations. Initialactivation steps in the absence of calcium can lead to magnifiedactivation.

In some embodiments, the host T-lymphocytes are modified to reduceapoptosis mediated killing of target cells by CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side-CAR T-cells. For example, the host T-cells canbe genetically modified to knock-out FasL which will prevent the CAR,Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR T-cell from killingtarget cells by FasL mediated apoptosis. In some embodiments, FasL isknocked out using the CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, orequivalent systems (e.g., Cong et al. Science 339.6121 (2013): 819-823,Li et al. Nucl. Acids Res (2011): gkr188, Gaj et al. Trends inBiotechnology 31.7 (2013): 397405, all of which are incorporated byreference in their entirety for all purposes). In some embodiments,double allele knockouts of the FasL gene can be obtained using a dualantibiotic resistant selection with Cas9 as described in Park et al.,PLoS One 9:e95101 (2014), which is incorporated by reference in itsentirety for all purposes, or the Cas9 gRNA approach as described inZhang et al., Methods 69:171-178 (2014), which is incorporated byreference in its entirety for all purposes. In some embodiments, doubleallele knock outs are obtained using a Cas9 system with multiple gRNAstargeted to the FasL gene of the host T-cell. These host T-lymphocyteswith a double knockout of FasL are then used as host cells for the CAR,Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side-CAR constructs of theinvention.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. In some embodiments,cells are enriched by cell sorting and/or selection via negativemagnetic immunoadherence or flow cytometry using a cocktail ofmonoclonal antibodies directed to cell surface markers present on thecells. For example, to enrich for CD4+ cells, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In some embodiments, it may be desirable to enrich forregulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+,and FoxP3+. Alternatively, in certain aspects, T regulatory cells aredepleted by anti-C25 conjugated beads or other similar method ofselection.

T cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005, each of which is incorporated by reference in its entiretyfor all purposes.

In some embodiments, NK cells may be expanded in the presence of amyeloid cell line that has been genetically modified to express membranebound IL-15 and 4-1BB ligand (CD137L). A cell line modified in this waywhich does not have MEW class I and II molecules is highly susceptibleto NK cell lysis and activates NK cells. For example, K562 myeloid cellscan be transduced with a chimeric protein construct consisting of humanIL-15 mature peptide fused to the signal peptide and transmembranedomain of human CD8a and GFP. Transduced cells can then be single-cellcloned by limiting dilution and a clone with the highest GFP expressionand surface IL-15 selected. This clone can then be transduced with humanCD137L, creating a K562-mb15-137L cell line. To preferentially expand NKcells, peripheral blood mononuclear cell cultures containing NK cellsare cultured with a K562-mb15-137L cell line in the presence of 10 IU/mLof IL-2 for a period of time sufficient to activate and enrich for apopulation of NK cells. This period can range from 2 to 20 days,preferably about 5 days. Expanded NK cells may then be transduced withthe anti-CD19-BB-ζ chimeric receptor.

Other host cells useful in the present invention are described in U.S.patent application Ser. No. 15/070,352 filed on Mar. 15, 2016, which isincorporated by reference in its entirety for all purposes.

Nucleic Acids

In some embodiments, the present invention relates to the nucleic acidsthat encode, at least in part, the individual peptides, polypeptides,proteins, and RNA control devices of the present invention. In someembodiments, the nucleic acids may be natural, synthetic or acombination thereof. The nucleic acids of the invention may be RNA,mRNA, DNA or cDNA.

In some embodiments, the nucleic acids of the invention also includeexpression vectors, such as plasmids, or viral vectors, or linearvectors, or vectors that integrate into chromosomal DNA. Expressionvectors can contain a nucleic acid sequence that enables the vector toreplicate in one or more selected host cells. Such sequences are wellknown for a variety of cells. The origin of replication from the plasmidpBR322 is suitable for most Gram-negative bacteria. In eukaryotic hostcells, e.g., mammalian cells, the expression vector can be integratedinto the host cell chromosome and then replicate with the hostchromosome. Similarly, vectors can be integrated into the chromosome ofprokaryotic cells.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.Generally, the selection gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. In some embodiments, anexemplary selection scheme utilizes a drug to arrest growth of a hostcell. Those cells that are successfully transformed with a heterologousgene produce a protein conferring drug resistance and thus survive theselection regimen. Other selectable markers for use in bacterial oreukaryotic (including mammalian) systems are well-known in the art.

An example of a promoter that is capable of expressing a Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR transgene in a mammalian T cell isthe EF1α promoter. The native EF1α promoter drives expression of thealpha subunit of the elongation factor-1 complex, which is responsiblefor the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1apromoter has been extensively used in mammalian expression plasmids andhas been shown to be effective in driving CAR expression from transgenescloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther.17(8): 1453-1464 (2009), which is incorporated by reference in itsentirety for all purposes. Another example of a promoter is theimmediate early cytomegalovirus (CMV) promoter sequence. This promotersequence is a strong constitutive promoter sequence capable of drivinghigh levels of expression of any polynucleotide sequence operativelylinked thereto. Other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus promoter (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter,phosphoglycerate kinase (PGK) promoter, MND promoter (a syntheticpromoter that contains the U3 region of a modified MoMuLV LTR withmyeloproliferative sarcoma virus enhancer, see, e.g., Li et al., J.Neurosci. Methods vol. 189, pp. 56-64 (2010) which is incorporated byreference in its entirety for all purposes), an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1apromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention is not limited to the use of constitutivepromoters.

Inducible promoters are also contemplated as part of the invention.Examples of inducible promoters include, but are not limited to ametallothionein promoter, a glucocorticoid promoter, a progesteronepromoter, a tetracycline promoter, a c-fos promoter, the T-REx system ofThermoFisher which places expression from the human cytomegalovirusimmediate-early promoter under the control of tetracycline operator(s),and RheoSwitch promoters of Intrexon. Karzenowski, D. et al.,BioTechiques 39:191-196 (2005); Dai, X. et al., Protein Expr. Purif42:236-245 (2005); Palli, S. R. et al., Eur. J. Biochem. 270:1308-1515(2003); Dhadialla, T. S. et al., Annual Rev. Entomol. 43:545-569 (1998);Kumar, M. B, et al., J. Biol. Chem. 279:27211-27218 (2004); Verhaegent,M. et al., Annal. Chem. 74:4378-4385 (2002); Katalam, A. K., et al.,Molecular Therapy 13:S103 (2006); and Karzenowski, D. et al., MolecularTherapy 13:S194 (2006), U.S. Pat. Nos. 8,895,306, 8,822,754, 8,748,125,8,536,354, all of which are incorporated by reference in their entiretyfor all purposes.

Expression vectors of the invention typically have promoter elements,e.g., enhancers, to regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the thymidine kinase (tk) promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either cooperatively or independentlyto activate transcription.

In some embodiments, it may be desirable to modify the polypeptides ofthe present invention. One of skill will recognize many ways ofgenerating alterations in a given nucleic acid construct to generatevariant polypeptides Such well-known methods include site-directedmutagenesis, PCR amplification using degenerate oligonucleotides,exposure of cells containing the nucleic acid to mutagenic agents orradiation, chemical synthesis of a desired oligonucleotide (e.g., inconjunction with ligation and/or cloning to generate large nucleicacids) and other well-known techniques (see, e.g., Gillam and Smith,Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734, 1987, which isincorporated by reference in its entirety for all purposes). In someembodiments, the recombinant nucleic acids encoding the polypeptides ofthe invention are modified to provide preferred codons which enhancetranslation of the nucleic acid in a selected organism.

The polynucleotides of the invention also include polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides of the invention. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide of the invention. The invention also provides thecomplement of the polynucleotides including a nucleotide sequence thathas at least about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotideencoding a polypeptide recited above. The polynucleotide can be DNA(genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithmsfor obtaining such polynucleotides are well known to those of skill inthe art and can include, for example, methods for determininghybridization conditions which can routinely isolate polynucleotides ofthe desired sequence identities.

Nucleic acids which encode protein analogs or variants in accordancewith this invention (i.e., wherein one or more amino acids are designedto differ from the wild type polypeptide) may be produced using sitedirected mutagenesis or PCR amplification in which the primer(s) havethe desired point mutations. For a detailed description of suitablemutagenesis techniques, see Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and/or Current Protocols in Molecular Biology,Ausubel et al., eds, Green Publishers Inc. and Wiley and Sons, N.Y.(1994), each of which is incorporated by reference in its entirety forall purposes. Chemical synthesis using methods well known in the art,such as that described by Engels et al., Angew Chem Intl Ed. 28:716-34,1989 (which is incorporated by reference in its entirety for allpurposes), may also be used to prepare such nucleic acids.

In some embodiments, amino acid “substitutions” for creating variantsare preferably the result of replacing one amino acid with another aminoacid having similar structural and/or chemical properties, i.e.,conservative amino acid replacements. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid.

The present invention provides a nucleic acid encoding the Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR of the invention. The nucleic acidencoding the Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR can beeasily prepared from an amino acid sequence of the specified CARcombined with the sequence of the RNA control device by a conventionalmethod. A base sequence encoding an amino acid sequence can be obtainedfrom the aforementioned NCBI Ref or accession numbers of GenBenk for anamino acid sequence of each element, and the nucleic acid of the presentinvention can be prepared using a standard molecular biological and/orchemical procedure. For example, based on the base sequence, a nucleicacid can be synthesized, and the nucleic acid of the present inventioncan be prepared by combining DNA fragments which are obtained from acDNA library using a polymerase chain reaction (PCR).

The nucleic acid of the present invention can be linked to anothernucleic acid so as to be expressed under control of a suitable promoter.The nucleic acid of the present invention can be also linked to, inorder to attain efficient transcription of the nucleic acid, otherregulatory elements that cooperate with a promoter or a transcriptioninitiation site, for example, a nucleic acid comprising an enhancersequence, a polyA site, or a terminator sequence. In addition to thenucleic acid of the present invention, a gene that can be a marker forconfirming expression of the nucleic acid (e.g. a drug resistance gene,a gene encoding a reporter enzyme, or a gene encoding a fluorescentprotein) may be incorporated.

When the nucleic acid of the present invention is introduced into a cellex vivo, the nucleic acid of the present invention may be combined witha substance that promotes transference of a nucleic acid into a cell,for example, a reagent for introducing a nucleic acid such as a liposomeor a cationic lipid, in addition to the aforementioned excipients.Alternatively, a vector carrying the nucleic acid of the presentinvention is also useful. Particularly, a composition in a form suitablefor administration to a living body which contains the nucleic acid ofthe present invention carried by a suitable vector is suitable for invivo gene therapy.

Reporters

In some embodiments, a reporter is a moiety capable of being detectedindirectly or directly. In some embodiments, the signal from a reportercan be used to quantify an aspect of the system containing the reporter.In some embodiments, reporters include, for example, a chromophore, afluorophore, a bioluminescent protein, a fluorescent protein, areceptor, a hapten, an enzyme, and a radioisotope. In some embodiments,a reporter is encoded by a reporter gene which polynucleotide encodes areporter molecule that can be detected, either directly or indirectly.In some embodiments, a reporter probe detects the presence (e.g.,expression) of a reporter molecule. The detectable label on the reporterprobe can be any detectable moiety, including, without limitation, anisotope, chromophore, and fluorophore. A reporter probe can be anydetectable molecule or composition that binds to or is acted upon by thereporter to permit detection of the reporter molecule. In someembodiments, the reporter is a fluorescent reporter, a bioluminescentreporter, other optical reporter, a radioactive reporter, a PositronEmission Tomography (PET) reporter, a Single Photon Emission ComputedTomography (SPECT) reporter, an X-Ray reporter, a photoacousticreporter, and an ultrasound reporter.

In some embodiments, reporter genes encode, for example, enzymes,fluorescent proteins, bioluminescent proteins, receptors, antigenicepitopes, or transporters. In some embodiments, the enzymes include, forexample, β-galactosidase, alkaline phosphatase, chloramphenicolacetyltransferase, horseradish peroxidase, or β-lactamase. In someembodiments, substrates for β-galactosidase include, for example, ONPG(o-nitrophenyl-β-D-galactopyranoside), Galacton-Plus®, Galacton-Star®,which are commercially available from ThermoFisher Scientific. In someembodiments, substrates for alkaline phosphatase include, for example,PNPP (p-Nitrophenyl Phosphate, Disodium Salt), CSPD® chemiluminescentsubstrate, 1,2-di oxetane chemiluminescent substrate, DynaLight™Substrate with RapidGlow™ Enhancer, which are all commercially availablefrom ThermoFisher Scientific. In some embodiments, substrates forchloramphenicol acetyltransferase include, for example, FAST CAT® Green(deoxy), which is commercially available from ThermoFisher Scientific.In some embodiments, substrates for horseradish peroxidase include, forexample, ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt), OPD (o-phenylenediamine dihydrochloride), TMB(3,3′,5,5′-tetramethylbenzidine), SuperSignal ELISA PicoChemiluminescent Substrate, QuantaBlu NS/K Fluorogenic Substrate,QuantaRed Enhanced Chemifluorescent HRP Substrate (ADHP), Amplex Redreagent, all of which are commercially available from ThermoFisherScientific. In some embodiments, substrates for β-lactamase include, forexample, CCF2-FA, CCF2-AM, CCF4-AM, Fluorocillin™ Green reagent,LyticBLAzer™_h-BODIPY® FL Substrate, which is commercially availablefrom ThermoFisher Scientific. Many other enzymes and substrates arewell-known in the art and can be used as reporter systems for theinvention.

In some embodiments, the fluorescent reporter includes, for example,green fluorescent protein, cyan fluorescent protein, yellow fluorescentprotein, orange fluorescent protein, red fluorescent protein, andfar-red fluorescent protein, all of which are commercially availablefrom Clontech. In some embodiments, the bioluminescent reporter include,for example, North American firefly luciferase, Japanese fireflyluciferase, Italian firefly luciferase, East European fireflyluciferase, Pennsylvania firefly luciferase, Click beetle luciferase,railroad worm luciferase, Renilla luciferase, Gaussia luciferase,Cypridina luciferase, Metrida luciferase, OLuc, and red fireflyluciferase, all of which are commercially available from ThermoFisherScientific and/or Promega.

In some embodiments, a reporter includes another agent that has adetectable moiety. In some embodiments, the reporter includes anantibody that is labeled with a detectable moiety. In some embodiments,the detectable moiety on the antibody can be an enzyme, a dye (e.g.,fluorescein (FITC), phycoerythrin (PE), Cy5PE, Cy7PE, Texas Red (TR),allophycocyanin (APC), Cy5, Cy7APC, cascade blue, Alexa Fluor®, CyDye®,IRDye®, DyLight, ATTO-TEC fluorescent labels, cyanine dyes, rhodaminedyes, fluorescent TrueBlot), a radioisotope, biotin, or haptens. Theseand other detectable moieties are well known in the art and many arecommercially available. In some embodiments, the reporter is aligand-receptor pair, and is detected by using one member of the pairlabeled with a detectable moiety.

In some embodiments, the reporter is detected by optical imaging,ultrasound imaging, computed tomography imaging, magnetic resonanceimaging, optical coherence tomography imaging, radiography imaging,nuclear medical imaging, positron emission tomography imaging,tomography imaging, photo acoustic tomography imaging, x-ray imaging,thermal imaging, fluoroscopy imaging, bioluminescent imaging, andfluorescent imaging, magnetic particle imaging, and magnetic resonancespectroscopy.

Process for Producing Eukaryotic Cells Expressing Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR

A process for producing a cell expressing the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR of the present invention includes a stepof introducing the nucleic acid encoding a Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR described above into a eukaryotic cell. Insome embodiments, this step is carried out ex vivo. For example, a cellcan be transformed ex vivo with a virus vector or a non-virus vectorcarrying the nucleic acid of the present invention to produce a cellexpressing the Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR of thepresent invention.

In the process of the present invention, a eukaryotic cell as describeabove is used. In some embodiments, a eukaryotic cell derived from amammal, for example, a human cell, or a cell derived from a non-humanmammal such as a monkey, a mouse, a rat, a pig, a horse, or a dog can beused. The cell used in the process of the present invention is notparticularly limited, and any cell can be used. For example, a cellcollected, isolated, purified or induced from a body fluid, a tissue oran organ such as blood (peripheral blood, umbilical cord blood etc.) orbone marrow can be used. A peripheral blood mononuclear cell (PBMC), animmune cell, a dendritic cell, a B cell, a hematopoietic stem cell, amacrophage, a monocyte, a NK cell or a hematopoietic cell, an umbilicalcord blood mononuclear cell, a fibroblast, a precursor adipocyte, ahepatocyte, a skin keratinocyte, a mesenchymal stem cell, an adiposestem cell, various cancer cell strains, or a neural stem cell can beused. In the present invention, particularly, use of a T cell, aprecursor cell of a T cell (a hematopoietic stem cell, a lymphocyteprecursor cell etc.) or a cell population containing them is preferable.Examples of the T cell include a CD8-positive T cell, a CD4-positive Tcell, a regulatory T cell, a cytotoxic T cell, and a tumor infiltratinglymphocyte. The cell population containing a T cell and a precursor cellof a T cell includes a PBMC. The aforementioned cells may be collectedfrom a living body, obtained by expansion culture of a cell collectedfrom a living body, or established as a cell strain. Whentransplantation of the produced Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR expressing cell or a cell differentiated from the producedSmart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR expressing cell into aliving body is desired, it is preferable to introduce the nucleic acidinto a cell collected from the living body itself.

In some embodiments, the nucleic acid encoding the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR of the present invention is inserted intoa vector, and the vector is introduced into a cell. In some embodiments,the nucleic acid encoding the Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR is introduced to the eukaryotic cell by transfection (e.g.,Gorman, et al. Proc. Natl. Acad. Sci. 79.22 (1982): 6777-6781, which isincorporated by reference in its entirety for all purposes),transduction (e.g., Cepko and Pear (2001) Current Protocols in MolecularBiology unit 9.9; DOI: 10.1002/0471142727.mb0909s36, which isincorporated by reference in its entirety for all purposes), calciumphosphate transformation (e.g., Kingston, Chen and Okayama (2001)Current Protocols in Molecular Biology Appendix 1C; DOI:10.1002/0471142301.nsa01cs01, which is incorporated by reference in itsentirety for all purposes), cell-penetrating peptides (e.g., Copolovici,Langel, Eriste, and Langel (2014) ACS Nano 2014 8 (3), 1972-1994; DOI:10.1021/nn4057269, which is incorporated by reference in its entiretyfor all purposes), electroporation (e.g. Potter (2001) Current Protocolsin Molecular Biology unit 10.15; DOI: 10.1002/0471142735.im1015s03 andKim et al (2014) Genome 1012-19. doi:10.1101/gr.171322.113, Kim et al.2014 describe the Amaza Nucleofector, an optimized electroporationsystem, both of these references are incorporated by reference in theirentirety for all purposes), microinjection (e.g., McNeil (2001) CurrentProtocols in Cell Biology unit 20.1; DOI: 10.1002/0471143030.cb2001s18,which is incorporated by reference in its entirety for all purposes),liposome or cell fusion (e.g., Hawley-Nelson and Ciccarone (2001)Current Protocols in Neuroscience Appendix 1F; DOI:10.1002/0471142301.nsa01fs10, which is incorporated by reference in itsentirety for all purposes), mechanical manipulation (e.g. Sharon et al.(2013) PNAS 2013 110(6); DOI: 10.1073/pnas.1218705110, which isincorporated by reference in its entirety for all purposes) or otherwell-known technique for delivery of nucleic acids to eukaryotic cells.Once introduced, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARnucleic acid can be transiently expressed episomally, or can beintegrated into the genome of the eukaryotic cell using well knowntechniques such as recombination (e.g., Lisby and Rothstein (2015) ColdSpring Harb Perspect Biol. March 2; 7(3). pii: a016535. doi:10.1101/cshperspect.a016535, which is incorporated by reference in itsentirety for all purposes), or non-homologous integration (e.g., Deyleand Russell (2009) Curr Opin Mol Ther. 2009 August; 11(4):442-7, whichis incorporated by reference in its entirety for all purposes). Theefficiency of homologous and non-homologous recombination can befacilitated by genome editing technologies that introduce targeteddouble-stranded breaks (DSB). Examples of DSB-generating technologiesare CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, or equivalent systems(e.g., Cong et al. Science 339.6121 (2013): 819-823, Li et al. Nucl.Acids Res (2011): gkr188, Gaj et al. Trends in Biotechnology 31.7(2013): 397-405, all of which are incorporated by reference in theirentirety for all purposes), transposons such as Sleeping Beauty (e.g.,Singh et al (2014) Immunol Rev. 2014 January; 257(1):181-90. doi:10.1111/imr.12137, which is incorporated by reference in its entiretyfor all purposes), targeted recombination using, for example, FLPrecombinase (e.g., O'Gorman, Fox and Wahl Science (1991)15:251(4999):1351-1355, which is incorporated by reference in itsentirety for all purposes), CRE-LOX (e.g., Sauer and Henderson PNAS(1988): 85; 5166-5170), or equivalent systems, or other techniques knownin the art for integrating the nucleic acid encoding the Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR into the eukaryotic cell genome.

In an embodiment, the nucleic acid encoding the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR is integrated into the eukaryotic cellchromosome at a genomic safe harbor site, such as, for example, theCCR5, AAVS1, human ROSA26, or PSIP1 loci. (Sadelain et al., Nature Rev.12:51-58 (2012); Fadel et al., J. Virol. 88(17):9704-9717 (2014); Ye etal., PNAS 111(26):9591-9596 (2014), all of which are incorporated byreference in their entirety for all purposes.) In an embodiment, theintegration of the nucleic acid encoding the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR at the CCR5 or PSIP1 locus is done using agene editing system, such as, for example, CRISPR, TALEN, or Zinc-Fingernuclease systems. In an embodiment, the eukaryotic cell is a human,T-lymphocyte and a CRISPR system is used to integrate the Smart CAR,DE-CAR, Smart-DE-CAR, and/or Side CAR at the CCR5 or PSIP1 locus. In anembodiment, integration of the nucleic acid at CCR5 or PSIP1 using theCRISPR system also deletes a portion, or all, of the CCR5 gene or PSIP1gene. In an embodiment, Cas9 in the eukaryotic cell may be derived froma plasmid encoding Cas9, an exogenous mRNA encoding Cas9, or recombinantCas9 polypeptide alone or in a ribonucleoprotein complex. (Kim et al(2014) Genome 1012-19. doi:10.1101/gr.171322.113; Wang et al (2013) Cell153 (4). Elsevier Inc.: 910-18. doi:10.1016/j.cell.2013.04.025, both ofwhich are incorporated by reference in their entirety for all purposes.)

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In some embodiments, transduction can be done with a virus vector suchas a retrovirus vector (including an oncoretrovirus vector, a lentivirusvector, and a pseudo type vector), an adenovirus vector, anadeno-associated virus (AAV) vector, a simian virus vector, a vacciniavirus vector or a sendai virus vector, an Epstein-Barr virus (EBV)vector, and a HSV vector can be used. As the virus vector, a virusvector lacking the replicating ability so as not to self-replicate in aninfected cell is preferably used.

In some embodiments, when a retrovirus vector is used, the process ofthe present invention can be carried out by selecting a suitablepackaging cell based on a LTR sequence and a packaging signal sequencepossessed by the vector and preparing a retrovirus particle using thepackaging cell. Examples of the packaging cell include PG13 (ATCCCRL-10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12 (U.S. Pat.No. 5,278,056, which is incorporated by reference in its entirety forall purposes), and Psi-Crip (Proceedings of the National Academy ofSciences of the United States of America, vol. 85, pp. 6460-6464 (1988),which is incorporated by reference in its entirety for all purposes). Aretrovirus particle can also be prepared using a 293 cell or a T cellhaving high transfection efficiency. Many kinds of retrovirus vectorsproduced based on retroviruses and packaging cells that can be used forpackaging of the retrovirus vectors are widely commercially availablefrom many companies.

A number of viral based systems have been developed for gene transferinto mammalian cells. A selected gene can be inserted into a vector andpackaged in viral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of viral systems are knownin the art. In some embodiments, adenovirus vectors are used. A numberof adenovirus vectors are known in the art. In some embodiments,lentivirus vectors are used.

In some embodiments, a viral vector derived from a RNA virus is used tointroduce the Smart CAR, Smart-DE-CAR, and/or Side-CAR encodingpolynucleotides. In some embodiments, the RNA virus vector encodes thereverse complement or antisense strand of the polynucleotide encodingthe RNA control device and CAR construct (the complementary strandencodes the sense strand for the RNA control device, DE, CAR and/orSide-CAR construct). In this embodiment, the RNA control device is notactive in the single stranded, RNA virus vector. In some embodiments,the sense strand of the RNA control device, DE, CAR and/or Side-CARconstruct is encoded in the RNA virus vector, and the viral vector withthe RNA control device, DE, CAR and/or Side-CAR construct is maintainedand replicated in the presence of ligand for the sensor element of theRNA control device. In some embodiments, the viral vector encoding thesense strand of the RNA control device, DE, CAR and/or Side-CARconstruct in the viral vector is maintained and replicated withoutligand for the sensor element.

In some embodiments, a non-virus vector is used in combination with aliposome and a condensing agent such as a cationic lipid as described inWO 96/10038, WO 97/18185, WO 97/25329, WO 97/30170 and WO 97/31934(which are incorporated herein by reference in their entirety for allpurposes). The nucleic acid of the present invention can be introducedinto a cell by calcium phosphate transduction, DEAE-dextran,electroporation, or particle bombardment.

In some embodiments, chemical structures with the ability to promotestability and/or translation efficiency are used. The RNA preferably has5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. In some embodiments, UTRsequences that are not endogenous to the nucleic acid of interest can beadded by incorporating the UTR sequences into the forward and reverseprimers or by any other modifications of the template. The use of UTRsequences that are not endogenous to the nucleic acid of interest can beuseful for modifying the stability and/or translation efficiency of theRNA. For example, it is known that AU-rich elements in 3′UTR sequencescan decrease the stability of mRNA. Therefore, 3′ UTRs can be selectedor designed to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In some embodiments, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

In the step of introducing a nucleic acid into a cell, a functionalsubstance for improving the introduction efficiency can also be used(e.g. WO 95/26200 and WO 00/01836, which are incorporated herein byreference in their entirety for all purposes). Examples of the substancefor improving the introduction efficiency include a substance havingability to bind to a virus vector, for example, fibronectin and afibronectin fragment. In some embodiments, a fibronectin fragment havinga heparin binding site, for example, a fragment commercially availableas RetroNetcin (registered trademark, CH-296, manufactured by TAKARA BIOINC.) can be used. Also, polybrene which is a synthetic polycationhaving an effect of improving the efficiency of infection of aretrovirus into a cell, a fibroblast growth factor, V type collagen,polylysine or DEAE-dextran can be used.

In a preferable aspect of the present invention, the functionalsubstance can be used in a state of being immobilized on a suitablesolid phase, for example, a container used for cell culture (plate,petri dish, flask or bag) or a carrier (microbeads etc.).

Eukaryotic Cells Expressing the Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR

The cell expressing the Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARof the present invention is a cell in which a nucleic acid encoding aSmart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR is introduced andexpressed.

In some embodiments, a eukaryotic cell of the present invention binds toa specific antigen via the CAR, DE-CAR, and/or Side-CAR polypeptidecausing the CAR, DE-CAR, and/or Side-CAR polypeptide to transmit asignal into the eukaryotic cell, and as a result, the eukaryotic cell isactivated. The activation of the eukaryotic cell expressing the SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR is varied depending on thekind of a eukaryotic cell and the intracellular element of the SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR, and can be confirmed basedon, for example, release of a cytokine, improvement of a cellproliferation rate, change in a cell surface molecule, or the like as anindex.

In some embodiments, eukaryotic cells expressing CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR constructs are detected using Protein L (abacterial surface protein isolated from Peptostreptoccocus magnus thatselectively binds to variable light chains (kappa chain) ofimmunoglobulins. In some embodiments, Protein L is directly labeled witha reporter (e.g., a light emitting or absorbing moiety) or is labeledwith an agent such as biotin. When biotin or related molecule is used tolabel the Protein L, binding of Protein L to eukaryotic cells displayingCAR, DE-CAR, and/or Side-CAR polypeptide is detected by adding astreptavidin (or similar paired molecule) labeled with reporter (e.g.,phycoerythrin). Zheng et al., J. Translational Med., 10:29 (2012), whichis incorporated by reference in its entirety for all purposes. Protein Lbinding to eukaryotic cells containing CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR constructs demonstrates the presence ofantibody light chain, the extracellular domain of a CAR, on theeukaryotic cell. This method of detecting CAR expression on theeukaryotic cell can also be used to quantitate the amount of CAR,DE-CAR, and/or Side-CAR polypeptide on the surface of the eukaryoticcell. In some embodiments, Protein L is used in QC and QA methodologiesfor making eukaryotic cells with the CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR constructs of the invention.

In some embodiments, a eukaryotic cell expressing the Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR is used as a therapeutic agent to treat adisease. The therapeutic agent comprises the eukaryotic cell expressingthe Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR as an activeingredient, and may further comprise a suitable excipient. Examples ofthe excipient include pharmaceutically acceptable excipients for thecomposition. The disease against which the eukaryotic cell expressingthe Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR is administered isnot particularly limited as long as the disease shows sensitivity to theeukaryotic cell. Examples of diseases of the invention include a cancer(blood cancer (leukemia), solid tumor etc.), hepatitis, or otherinfectious disease the cause of which is a virus such as influenza andHIV, a bacterium, or a fungus, for example, tuberculosis, MRSA, VRE, anddeep mycosis. Other diseases that may be treated with eukaryotic cellsexpressing the Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR aredisclosed in U.S. patent application Ser. No. 15/070,352 filed on Mar.15, 2016, which is incorporated by reference in its entirety for allpurposes.

The eukaryotic cell expressing the Smart CAR, DE-CAR, Smart-DE-CAR,and/or Side CAR of the present invention is administered for treatmentof these diseases. The eukaryotic cell of the present invention can alsobe utilized for prevention of an infectious disease after bone marrowtransplantation or exposure to radiation, donor lymphocyte transfusionfor the purpose of remission of recurrent leukemia, and the like. Thetherapeutic agent comprising the eukaryotic cell expressing the SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR as an active ingredient canbe administered intradermally, intramuscularly, subcutaneously,intraperitoneally, intranasally, intraarterially, intravenously,intratumorally, or into an afferent lymph vessel, by parenteraladministration, for example, by injection or infusion, although theadministration route is not limited.

In some embodiments, the eukaryotic cells with Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR are characterized prior to administrationto the subject. In some embodiments, the eukaryotic cells with SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR are tested to confirm SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR expression. In someembodiments, the eukaryotic cells with Smart CAR, DE-CAR, Smart-DE-CAR,and/or Side CAR are exposed to a level of ligand(s) that results in adesired level of CAR, DE-CAR, and/or Side-CAR polypeptide expression inthe eukaryotic cell. In some embodiments, this desired level of CAR,DE-CAR, and/or Side-CAR polypeptide produces eukaryotic cells with adesired level of anti-target cell activity, and/or a desired level ofproliferative activity when placed in a subject.

In some embodiments, the Smart CAR, DE-CAR, Smart-DE-CAR, and/or SideCAR is used with a T-lymphocyte that has aggressive anti-tumorproperties, such as those described in Pegram et al, CD28z CARs andarmored CARs, 2014, Cancer J. 20(2):127-133, which is incorporated byreference in its entirety for all purposes. In some embodiments, the RNAcontrol device of the invention is used with an armored CAR, DE-CAR,and/or Side-CAR polypeptide in a T-lymphocyte.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise aSmart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR expressing cell, e.g.,a plurality of Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARexpressing cells, as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arein one aspect formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

Suitable pharmaceutically acceptable excipients are well known to aperson skilled in the art. Examples of the pharmaceutically acceptableexcipients include phosphate buffered saline (e.g. 0.01 M phosphate,0.138 M NaCl, 0.0027 M KCl, pH 7.4), an aqueous solution containing amineral acid salt such as a hydrochloride, a hydrobromide, a phosphate,or a sulfate, saline, a solution of glycol or ethanol, and a salt of anorganic acid such as an acetate, a propionate, a malonate or a benzoate.In some embodiments, an adjuvant such as a wetting agent or anemulsifier, and a pH buffering agent can also be used. In someembodiments, the pharmaceutically acceptable excipients described inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) (which isincorporated herein by reference in its entirety for all purposes) canbe appropriately used. The composition of the present invention can beformulated into a known form suitable for parenteral administration, forexample, injection or infusion. In some embodiments, the composition ofthe present invention may comprise formulation additives such as asuspending agent, a preservative, a stabilizer and/or a dispersant, anda preservation agent for extending a validity term during storage.

A composition comprising the eukaryotic cells of the present inventionas an active ingredient can be administered for treatment of, forexample, a cancer (blood cancer (leukemia), solid tumor etc.), aninflammatory disease/autoimmune disease (asthma, eczema), hepatitis, andan infectious disease the cause of which is a virus such as influenzaand HIV, a bacterium, or a fungus, for example, a disease such astuberculosis, MRSA, VRE, or deep mycosis, depending on an antigen towhich a CAR, DE-CAR, and/or Side-CAR polypeptide binds.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intranasally, intraarterially,intratumorally, into an afferent lymph vessel, by intravenous (i.v.)injection, or intraperitoneally. In one aspect, the T cell compositionsof the present invention are administered to a patient by intradermal orsubcutaneous injection. In one aspect, the T-cell compositions of thepresent invention are administered by i.v. injection. The compositionsof T-cells may be injected directly into a tumor, lymph node, or site ofinfection. In some embodiments, the administration is adoptive transfer.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). In some embodiments, a pharmaceutical composition comprisingthe eukaryotic cells described herein may be administered at a dosage of10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ cells/kgbody weight, including all integer values within those ranges. In someembodiments, a eukaryotic cell composition may also be administeredmultiple times at these dosages. In some embodiments, eukaryotic cellscan be administered by using infusion techniques that are commonly knownin immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988, which is incorporated by reference in its entirety forall purposes).

Identification of Antigen Binding Domains and CAR Constructs forTreatment of Certain Diseases and Conditions

In some embodiments, antigen binding domains and CAR constructs withthose antigen binding domains are made from lymphocytes obtained from asubject that has generated an immune response against a disease orcondition. In some embodiments, antigen binding domains and CARconstructs with those antigen binding domains are made from lymphocytesobtained from a subject that has generated an effective immune responseagainst a disease or condition. In some embodiments, the antigen bindingdomains obtained from the subject with immunity are antigen bindingdomains capable of neutralizing the causative agent of the disease orcondition. In some embodiments, the disease is an infectious diseasecaused by a pathogen. In some embodiments, the pathogen is a virus, abacterium, or a fungus. In some embodiments, the disease is a cancer. Insome embodiments, the condition is an autoimmune disorder. In someembodiments, lymphocytes from the subject are obtained. In someembodiments, the lymphocytes include, for example, B-cells, memoryB-cells, plasma cells, and/or pre-B-cells. In some embodiments,lymphocytes include, for example, T-cells, Th1 T-cells, Th2 T-cells,Th17 T-cells, other helper T-cells, Treg T-cells, cytotoxic T-cells,and/or memory T-cells. In some embodiments, lymphocytes are obtainedfrom the peripheral blood of a subject. In some embodiments, thelymphocytes are obtained from the spleen and/or lymph nodes of asubject. In some embodiments, the lymphocytes are obtained from the bonemarrow of a subject. In some embodiments, the lymphocytes are obtainedfrom one of more of the peripheral blood, spleen, lymph nodes, and/orbone marrow of a subject. In some embodiments, the lymphocytes areobtained from a mammal. In some embodiments, the lymphocytes areobtained from a mouse. In some embodiments, the lymphocytes are obtainedfrom a rabbit. In some embodiments, the lymphocytes are obtained from ahuman.

In some embodiments, B-cells and/or B-cell subpopulations (memoryB-cells, plasma cells) are isolated from the lymphocytes usingtechniques known to a person or ordinary skill in the art including, forexample, commercially available kits from STEMCELL Technologies, Inc.,Miltenyi Biotec, Inc., and Thermo Fisher Scientific, Inc. In someembodiments, T-cells and T-cell subpopulations (memory T-cells, CD8+T-cells, CD4+ T-cells) are isolated from lymphocytes using techniquesknown to a person or ordinary skill in the art including, for example,commercially available kits from STEMCELL Technologies, Inc., MiltenyiBiotec, Inc., and Thermo Fisher Scientific, Inc.

In some embodiments, B-cells or a subpopulation of B-cells from asubject(s) are used to make a library of antigen binding domains. Insome embodiments, nucleic acids encoding the light and heavy chain ofthe antigen binding domain are amplified from either (or both) thegenomic DNA of the B-cells (or subpopulation of B-cells) and/or the mRNAof the B-cells (or subpopulation of B-cells). Techniques and primers foramplifying nucleic acids encoding human antibody light and heavy chainsare well-known in the art, and described in, for example, ProGen's HumanIgG and IgM Library Primer Set, Catalog No. F2000; Andris-Widhopf etal., “Generation of Human Fab Antibody Libraries: PCR Amplification andAssembly of Light and Heavy Chain Coding Sequences,” Cold Spring Harb.Protoc. 2011; Lim et al., Nat. Biotechnol. 31:108-117 (2010); Sun etal., World J. Microbiol. Biotechnol. 28:381-386 (2012); Coronella etal., Nucl. Acids. Res. 28:e85 (2000), all of which are incorporated byreference in their entirety for all purposes. Techniques and primers foramplifying nucleic acids encoding mouse antibody light and heavy chainsare well-known in the art, and described in, for example, U.S. Pat. No.8,143,007; Wang et al., BMC Bioinform. 7(Suppl):S9 (2006), both of whichare incorporated by reference in their entirety for all purposes. Insome embodiments, the antibody repertoires are used as separate chainsin antigen binding domains, or converted to single chain antigen bindingdomains. In some embodiments, single chain antibodies are made fromnucleic acids encoding human light and heavy chains using techniqueswell-known in the art including, for example, those described in Pansriet al., BMC Biotechnol. 9:6 (2009); Peraldi-Roux, Methods Molc. Biol.907:73-83 (2012), both of which are incorporated by reference in theirentirety for all purposes. In some embodiments, single chain antibodiesare made from nucleic acids encoding mouse light and heavy chains usingtechniques well-known in the art including, for example, those describedin Imai et al., Biol. Pharm. Bull. 29:1325-1330 (2006); Cheng et al.,PLoS ONE 6:e27406 (2011), both of which are incorporated by reference intheir entirety for all purposes.

In some embodiments, T-cells or a subpopulation of T-cells from asubject(s) are used to make a library of antigen binding domains. Insome embodiments, nucleic acids encoding T-cell receptors are amplifiedfrom either (or both) the genomic DNA of the T-cells (or subpopulationof T-cells) and/or the mRNA of the T-cells (or subpopulation ofT-cells). Techniques and primers for amplifying nucleic acids encodingthe T-cell receptors from lymphocytes are well known in the art and aredescribed in, for example, SMARTer Human TCR a/b Profiling Kits soldcommercially by Clontech, Boria et al., BMC Immunol. 9:50-58 (2008);Moonka et al., J. Immunol. Methods 169:41-51 (1994); Kim et al., PLoSONE 7:e37338 (2012); Seitz et al., Proc. Natl Acad. Sci. 103:12057-62(2006), all of which are incorporated by reference in their entirety forall purposes. In some embodiments, the TCR repertoires are used asseparate chains to form an antigen binding domain. In some embodiments,the TCR repertoires are converted to single chain antigen bindingdomains. In some embodiments, single chain TCRs are made from nucleicacids encoding human alpha and beta chains using techniques well-knownin the art including, for example, those described in U.S. PatentApplication Publication No. US2012/0252742, Schodin et al., Mol.Immunol. 33:819-829 (1996); Aggen et al., “Engineering HumanSingle-Chain T Cell Receptors,” Ph.D. Thesis with the University ofIllinois at Urbana-Champaign (2010) a copy of which is found atideals.illinois.edu/bitstream/handle/2142/18585/AggenDavid.pdf?sequence=1, all of which are incorporated by reference intheir entirety for all purposes.

In some embodiments, natural killer cells, dendritic cells, macrophages,T-cells, and/or B-cells are used to make a library of NKG receptorbinding domains and/or Toll-like receptor binding domains. In someembodiments, the natural killer cells, dendritic cells, macrophages,T-cells, and/or B-cells are obtained from a subject who has becomeimmune to a disease or has had an immune response to a disease orcondition. In some embodiments, the antigen binding domains from theCD94/NKG2 receptor family (e.g., NKG2A, NKG2B, NKG2C, NKG2D, NKG2E,NKG2F, NKG2H), the 2B4 receptor, the NKp30, NKp44, NKp46, and NKp80receptors, the Toll-like receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, RP105), and/or innate immunity receptorsare obtained from the subjects immune cells (natural killer cells,dendritic cells, macrophages, T-cells, and B-cells). In someembodiments, the extracellular elements for the library are made asdescribed in U.S. Pat. Nos. 5,359,046, 5,686,281 and 6,103,521 (whichare hereby incorporated by reference in their entirety for allpurposes). In some embodiments, the extracellular element is part of areceptor which is monomeric, homodimeric, heterodimeric, or associatedwith a larger number of proteins in a non-covalent complex. In someembodiments, a multimeric receptor has only one polypeptide chain with amajor role in binding to the ligand. In these embodiments, theextracellular element can be derived from the polypeptide chain thatbinds the ligand. In some embodiments, the receptor is a complex ofextracellular portions from several proteins that forms covalent bondsthrough disulfide linkages. In this embodiment, the extracellularelement of the CAR may also form such multimeric extracellularcomplexes. In some embodiments, the extracellular element is comprisedof truncated portions of the receptor, where such truncated portion isfunctional for binding ligand.

In some embodiments, nucleic acids encoding the antibody repertoire fromB-cells or subpopulations of B-cells from a subject are used as theantigen binding domain for CAR chassis, Smart CAR chassis, DE-CARchassis, Smart-DE-CAR chassis, or Side CAR chassis. In some embodiments,nucleic acids encoding single chain antibodies obtained by combinatorialpairing of the nucleic acids encoding the light and heavy chains ofantibodies from the B-cells or subpopulations of B-cells from a subjectare combined with the CAR chassis, Smart CAR chassis, DE-CAR chassis,Smart-DE-CAR chassis, or Side CAR chassis. In some embodiments, thelibrary of CARs, Smart CARs, DE-CARs, Smart-DE-CARS, or Side CARs madewith the antibody repertoire are placed into eukaryotic cells (e.g.,lymphocytes) to make a library of eukaryotic cells with the library ofCARs, Smart CARs, DE-CARs, Smart-DE-CARS, or Side CARs.

In some embodiments, nucleic acids encoding the T-cell receptorrepertoire from T-cells or subpopulations of T-cells from a subject areused as the antigen binding domain for CAR chassis, Smart CAR chassis,DE-CAR chassis, Smart-DE-CAR chassis, or Side CAR chassis. In someembodiments, nucleic acids encoding single chain TCRs obtained bycombinatorial pairing of the nucleic acids encoding the alpha and betachains of TCRs from the T-cells or subpopulations of T-cells from asubject are combined with the CAR chassis, Smart CAR chassis, DE-CARchassis, Smart-DE-CAR chassis, or Side CAR chassis. In some embodiments,the library of CARs, Smart CARs, DE-CARs, Smart-DE-CARS, or Side CARsmade with the T-cell receptor repertoire are placed into eukaryoticcells (e.g., lymphocytes) to make a library of eukaryotic cells with thelibrary of CARs, Smart CARs, DE-CARs, Smart-DE-CARS, or Side CARs.

In some embodiments, this library of eukaryotic cells is challenged withan antigen and eukaryotic cells with a CAR, Smart CAR, DE-CAR,Smart-DE-CAR or Side CAR that have an antigen binding domain thatinteracts with the antigen can be stimulated to proliferate. In someembodiments, the antigen binding domains with the strongest binding tothe antigen will proliferate best, and so this challenge with antigenwill select for CARs, Smart CARs, DE-CARs, Smart-DE-CARS, or Side CARsthat bind to the antigen, and those CARs, Smart CARs, DE-CARs,Smart-DE-CARS, or Side CARs which bind strongly to the antigen mayproliferate best. In some embodiments, the antigen challenge producesantigen binding domains that will bind the antigen. In some embodiments,the antigen challenge is done with a tumor associated antigen and thisproduces CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARconstructs that can be used in a cancer and/or tumor therapy. In someembodiments, the antigen challenge is done with an antigen from aninfectious disease and this produces CAR, Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR constructs that can be used in therapiesto treat the infectious disease. In some embodiments, the antigenchallenge is done with an antigen from a cell infected with a virus andthis produces CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CARconstructs that can be used in therapies to treat infection by thevirus.

In some embodiments, eukaryotic cell further comprises a reporter thatproduces a detectable signal when the CAR, DE-CAR, and/or Side-CARpolypeptide is activated by antigen. In some embodiments, binding ofantigen by the CAR, DE-CAR, and/or Side-CAR polypeptide activates theT-cell and this activation induces expression of the reporter from areporter gene that is operably linked to an NFAT control region. In someembodiments, activation of the CAR produces a second messenger thatactivates a control region linked to a reporter gene or binds to a RNAcontrol device or DE that controls the expression of the reporter. Insome embodiments, binding of antigen by the CAR, DE-CAR, and/or Side-CARpolypeptide leads to the phosphorylation of other polypeptides in theeukaryotic cell. In some embodiments, the reporter is activated byphosphorylation. In some embodiments, the reporter is indirectlyactivated by phosphorylation of a polypeptide. In some embodiments, thesignal produced from the reporter is used as a measure of antigeninteraction with the antigen binding domain, and for example, positivereporter signal may be used to identify antigen binding domains that canbind to an antigen. In some embodiments, the reporter is an opticalreporter (e.g., a bioluminescent protein such as luciferase or afluorescent protein such as GFP) and eukaryotic cells showing an opticalsignal above a threshold amount (indicating a threshold degree ofbinding of antigen by the antigen binding domain) are identified. Insome embodiments, the threshold amount of optical activity is measuredby a fluorescence activated cell sorter, such as those commercially soldby Becton Dickinson Biosciences, ThermoFisher, and Beckman Coulter. Insome embodiments, FACs is used to separate cells with antigen bindingdomains that interact with antigen from those cells that do not interactwith antigen.

In some embodiments, the CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR constructs obtained in the antigen challenge are challengedwith antigen for one or more cycles. In some embodiments, the subsequentchallenges with antigen further select for antigen binding domains thatbind to the antigen and/or further enriches for antigen binding domainswith higher affinity for the antigen. In some embodiments, ligand forthe Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR is/are changedduring subsequent challenges so that the amount of CAR, DE-CAR, and/oroperable Side CAR in the eukaryotic cells is decreased. In someembodiments, decreasing the amount of CAR, DE-CAR, and/or operable SideCAR in the cells can select for antigen binding domains having greateraffinity for the antigen.

In some embodiments, an antigen binding domain is given random ordirected changes in the CDRs and associated sequences and subsequentchallenges with antigen select and/or enrich for increased affinity inbinding to the antigen. In some embodiments, the ligand for the SmartCAR, DE-CAR, Smart-DE-CAR, and/or Side CAR is/are changed duringsubsequent challenges so that the amount of CAR, DE-CAR, and/or operableSide CAR in the eukaryotic cells is decreased. In some embodiments,decreasing the amount of CAR, DE-CAR, and/or operable Side CAR in thecells can select for antigen binding domains with greater affinity forthe antigen. In some embodiments, this affinity maturation of theantigen binding domains can increase the affinity for antigen by theantigen binding domain to a desired level.

In some embodiments, clones are obtained from the eukaryotic cells withthe library of CAR, Smart-CAR, DE-CAR, SMART-DE-CAR, and/or Side CARconstructs. In some embodiments, the nucleic acids encoding the antigenbinding domain form the clone are amplified or cut out of the nucleicacid encoding the CAR, Smart-CAR, DE-CAR, SMART-DE-CAR, and/or Side CARand cloned into a suitable recombinant vector for expression of theantigen binding domain. In some embodiments, the selected antigenbinding domains are sequenced. In some embodiments, the selected antigenbinding domains are expressed, purified and characterized for binding tothe target antigen. In some embodiments, these antigen binding domainsare used in therapies to treat the disease associated with the targetantigen.

Identification of Target Antigen for the New Antigen Binding Domains

In some embodiments, the new antigen binding domains are identifiedusing impure fractions or whole cells (or whole virus) as targets forthe CAR, Smart CAR, DE-CAR, Smart-DE-CAR, and/or Side CAR libraries. Insome embodiments, the whole cells may be diseased cells (e.g., cancercells or cells infected with a pathogen) or healthy cells. In someembodiments, techniques well known in the art are used to enrich and/orpurify the target antigen from the whole cells (or whole virus) orimpure fractions, including, for example, electrophoretic, molecular,immunological and chromatographic techniques, including ion exchange,hydrophobic, affinity, and reverse-phase HPLC chromatography, andchromatofocusing. Protein Purification: Principles and Practice,Springer Science and Business Media, 3^(rd) Edition (1994); MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); Ausubel et al.,Eds Short Protocols in Molecular Biology (5th Ed. 2002); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985), all of which are incorporatedby reference in their entirety for all purposes. In some embodiments,the antigen binding domain is used to purify the antigen using affinitytechniques, e.g., an antigen binding domain column for affinitychromatography, precipitation of the antigen from a sample using theantigen binding domain, or attachment of the antigen binding domain tobeads (e.g., magnetic beads) for separation of the antigen from asample.

In some embodiments, the fractions obtained from the purification stepsare tested for binding by the antigen binding domains. In someembodiments, binding by the antigen binding domain to the target antigenis detected by methods well known in the art, including, for example,FRET (Fluorescence Resonance Energy Transfer) and BRET (BioluminescenceResonance Energy Transfer)-based assays, AlphaScreen™ (AmplifiedLuminescent Proximity Homogeneous Assay), Scintillation Proximity Assay,ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface PlasmonResonance, also known as BIACORE®), isothermal titration calorimetry,differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration.

In some embodiments, the above methods provide pairs of antigen bindingdomains together with validated target antigens. In some embodiments,the target antigen is a polypeptide or a modified polypeptide (e.g.,glycosylated). In some embodiments, the polypeptide is subjected toanalysis to determine its amino acid composition, and its amino acidsequence using, for example, techniques and services availablecommercially from Bioproximity, LLC, Alphalyse, Inc., and AppliedBiomics, Inc. In some embodiments, the polypeptide sequence obtained forthe target antigen is used to identify the gene encoding the polypeptidefrom known gene sequences found at, for example, the National Center forBiotechnology Information. In some embodiments, the gene encoding thetarget antigen is not found in available databases, and the polypeptidesequence information is used to clone the gene encoding the targetantigen using techniques well known in the art including, for example,those taught in Maniatis (Cold Spring Harbor Laboratory Press: 1989);Ausubel et al., Eds Short Protocols in Molecular Biology (5th Ed. 2002),which are incorporated by reference in their entirety for all purposes.

In some embodiments, further validation is performed to show that theidentified target antigen is enriched on target cells compared tohealthy tissue in subjects. In some embodiments, the target antigens arecharacterized to show that a therapeutic effect is obtained when thesubject is treated with antigen binding domains (e.g., antibody drugconjugates) and/or cells with Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CARs specific for the target antigen.

Uses of Eukaryotic Cells with Smart CAR, DE-CAR, Smart-DE-CAR, and/orSide CAR

In some embodiments, nucleic acids encoding Smart CAR(s), DE-CAR(s),Smart-DE-CAR(s), and/or Side CAR(s) of the invention are used to expressCAR, DE-CAR, and/or Side-CAR polypeptides in eukaryotic cells. In someembodiments, nucleic acids encoding Smart CAR(s), DE-CAR(s),Smart-DE-CAR(s), and/or Side CAR(s) of the invention are used to expressCAR, DE-CAR, and/or Side-CAR polypeptides in mammalian cells. In someembodiments, nucleic acids encoding Smart CAR(s), DE-CAR(s),Smart-DE-CAR(s), and/or Side CAR(s) of the invention are used to expressCAR, DE-CAR, and/or Side-CAR polypeptides in human cells or murinecells. In some embodiments, nucleic acids encoding Smart CAR(s),DE-CAR(s), Smart-DE-CAR(s), and/or Side CAR(s) of the invention are usedto express CAR, DE-CAR, and/or Side-CAR polypeptide in hematopoieticcells. In some embodiments, nucleic acids encoding Smart CAR(s),DE-CAR(s), Smart-DE-CAR(s), and/or Side CAR(s) of the invention are usedto express CAR, DE-CAR, and/or Side-CAR polypeptides in T-cells, naturalkiller cells, B-cells, or macrophages. In some embodiments, nucleicacids encoding Smart CAR(s), DE-CAR(s), Smart-DE-CAR(s), and/or SideCAR(s) of the invention are used to express CAR, DE-CAR, and/or Side-CARpolypeptides in T-cells or natural killer cells.

In some embodiments, the nucleic acids encoding the Smart CAR(s),DE-CAR(s), Smart-DE-CAR(s), and/or Side CAR(s) of the invention are usedto express a desired level of CAR, DE-CAR, and/or Side-CAR polypeptideon the surface of the eukaryotic cell. In this embodiment, the DE, RNAcontrol device, and/or Side-CAR controls the level of CAR, DE-CAR,and/or Side-CAR polypeptide expression, at least in part, and bymodulating the level of activity of the DE, RNA control device, and/orSide-CAR, a desired amount of CAR, DE-CAR, and/or Side-CAR polypeptideis expressed and displayed on the surface of the eukaryotic cell. Insome embodiments, the DE increases the degradation rate of DE-CARpolypeptide in the eukaryotic cell and when ligand is bound by the DE,the rate of degradation decreases. In some embodiments, the DE increasesdegradation of the DE-CAR polypeptide when ligand is bound by the DE. Insome embodiments, the RNA control device inhibits translation of theDE-CAR mRNA and when ligand binds to the sensor element of the RNAcontrol device this inhibition of translation is reduced so that DE-CARpolypeptide expression is increased. In some embodiments, ligand for theSide-CAR causes the two Side-CAR polypeptides to form an active CAR. Insome embodiments, ligand for the DE, the ligand for the RNA controldevice sensor, and/or the ligand for the Side-CAR is added in increasingamounts to the eukaryotic cells with the Smart CAR(s), DE-CAR(s),Smart-DE-CAR(s), and/or Side CAR(s) until a desired level of CAR,DE-CAR, and/or Side-CAR polypeptide is made in the eukaryotic cell. Insome embodiments, the amount of CAR, DE-CAR, and/or Side-CAR polypeptideis measured using antibodies specific for the CAR, DE-CAR, and/orSide-CAR polypeptide. In some embodiments, the amount of CAR, DE-CAR,and/or Side-CAR polypeptide is measured using the antigen recognized bythe extracellular element. In some embodiments, the amount of CAR,DE-CAR, and/or Side-CAR polypeptide is measured in a functional assay oftarget cell killing. In some embodiments, the amount of CAR, DE-CAR,and/or Side-CAR polypeptide is measured in a functional assay foreukaryotic cell proliferation (induced by the CAR, DE-CAR, and/orSide-CAR polypeptide). In some embodiments, the above eukaryotic cell isa T-lymphocyte or a natural killer cell or a macrophage or otherphagocytic cell type.

In some embodiments, the ligand for the DE, the ligand for the RNAcontrol device sensor, and/or the ligand for the Side-CAR is added inincreasing amounts until a desired level of eukaryotic cell activity isobtained. In some embodiments, the desired eukaryotic cell activity iskilling of a target cell. In some embodiments, target cell killingoccurs over a desired time period, e.g., the killing of a certain numberof target cells in 12 hours, or 24 hours, or 36 hours, or two days, or3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, or 28 days, or two months, or 3, 4, 5, or 6 months.In some embodiments, target cell killing is expressed as a half-life fora standardized number of target cells. In this embodiment, the half-lifeof target cell killing can be 12 hours, 24 hours, 36 hours, or 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 days, or two months, or 3, 4, 5, or 6 months. Insome embodiments, the desired eukaryotic cell activity is proliferation.In some embodiments, the cell proliferation occurs with a doubling timeof 12 hours, 24 hours, 36 hours, two days, or 3, 4, 5, 6, or 7 days. Insome embodiments, the above eukaryotic cell is a T-lymphocyte or anatural killer cell or a macrophage or other phagocytic cell type.

In some embodiments, a regime of different amounts of ligand (for thesensor, DE, and/or Side-CAR) is added over time so that differentdesired levels of CAR, DE-CAR, and/or Side-CAR polypeptide are presenton the eukaryotic cell at different times. For example, during theenrichment/selection of antigen binding domains with Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR T-cells or Smart CAR, DE-CAR,Smart-DE-CAR, and/or Side CAR natural killer cells, the amount of CAR,DE-CAR, and/or Side-CAR polypeptide expression may be relatively highinitially to ensure that some antigen binding domains are able to bindtarget antigen, and in subsequent rounds of enrichment/selection, theamount of CAR, DE-CAR, and/or Side-CAR polypeptide expression can bedecreased to enrich/select for antigen binding domains with higheraffinity for target antigen. In some embodiments, the CAR, DE-CAR,and/or Side-CAR polypeptide expression may be relatively low initiallyto enrich/select for antigen binding domains that bind tightly to thetarget antigen, and in subsequent rounds CAR, DE-CAR, and/or Side-CARpolypeptide expression level is increased to increase the proliferationrate of the clones with antigen binding domains that bind targetantigen.

In some embodiments the nucleic acid sequences encoding a cognate RNAcontrol device or devices are present in a nucleic acid locus encoding achimeric antigen receptor transgene. In some embodiments, RNA controldevices are encoded for as nucleic acid sequence in the vector proximal,distal, or within the ORF encoding a CAR, DE-CAR, and/or Side-CARpolypeptide. An example of a schematic of a vector is included in FIG.1, adapted from (Budde et al., PLoS1, 2013,doi:10.1371/journal.pone.0082742, which is incorporated by reference inits entirety for all purposes). In some embodiments nucleic acidsequences encoding an RNA control device or devices are located withinthe 3′ UTR region of the transgene. In some embodiments nucleic acidsequences encoding an RNA control device or devices are located in the5′ UTR region of the DE-CAR transgene. In some embodiments nucleic acidsequences encoding an RNA control device or devices are located withinsynthetic or natural introns flanked by coding or noncoding exons withinthe CAR transgene, or at intron/exon boundaries.

Other uses of the CAR, Smart CAR, DE-CAR, Smart-DE-CAR, Side CAR and/oruniversal-CARs of the invention are described in U.S. patent applicationSer. No. 15/070,352 filed on Mar. 15, 2016, which is incorporated byreference in its entirety for all purposes. For example, U.S. Ser. No.15/070,352 describes the used of multiple TAA targets, multiple DEand/or RNA control devices for a CAR, and universal CARs. The use of RNAcontrol devices and/or DEs with other genes in eukaryotic cellsexpressing CAR, Smart CAR, DE-CAR, Smart-DE-CAR, Side CAR and/oruniversal-CARs of the invention is also described in U.S. patentapplication Ser. No. 15/070,352 filed on Mar. 15, 2016, which isincorporated by reference in its entirety for all purposes.

Target Cell Killing Assay

In some embodiments, methods are used to measure target cell killingafter the addition of a cell killing agent. In some embodiments, thecell killing agent is, for example, a host cell with a CAR device, SmartCAR device, DE CAR device, Smart-DE CAR device, and/or Side-CAR devices,a small molecule agent, a biological agent (e.g., an antibody, acytokine, an antibody-drug conjugate, etc.), a small molecule drugconjugate, or a nanoparticle-drug conjugate. In some embodiments, thetarget cell is modified to contain a reporter that is released when thetarget cell is killed. In some embodiments, the reporter is an opticalreporter, an enzyme (e.g., β-galactosidase, alkaline phosphatase,chloramphenicol acetyltransferase, horseradish peroxidase, β-lactamase),a fluorescent reporter (e.g., GFP, RFP), a bioluminescent reporter(e.g., luciferase), or another reporter. In some embodiments, the targetcell is a mammalian cell, a mouse cell, a rat cell, a human cell, abacterial cell, or a fungal cell. In some embodiments, the target cellis a diseased cell, such as for example, a cancer cell, or a cellinfected with a bacterial, fungal, and/or viral pathogen.

In some embodiments, a target cell killing assay is used for qualitycontrol and quality assurance in the making of a host cell containing aCAR device, Smart CAR device, DE CAR device, Smart-DE CAR device, and/orSide-CAR devices. In some embodiments, the target cell killing assay ofthe invention is used to define part of a specification for a host cellcontaining a CAR device, Smart CAR device, DE CAR device, Smart-DE CARdevice, and/or Side-CAR devices for the treatment of certain diseases.In some embodiments, the cell killing assay of the invention is used toengineer parts of a CAR device, Smart CAR device, DE CAR device,Smart-DE CAR device, and/or Side-CAR devices. In some embodiments, thecell killing assay of the invention is used in a screening assay toidentify clones with more or less activity.

In some embodiments, the target cell is made to express a reporter byengineering a reporter gene into the target cell. In some embodiments,the reporter gene is inserted into a safe harbor site of the targetcell. In some embodiments, the reporter gene is inserted into a desiredsite of the target cell that produces a phenotype in addition to thereporter phenotype. In some embodiments, the reporter gene istransiently engineered into the target cell (e.g., plasmid borne). Insome embodiments, the reporter gene is under the control of aconstitutive control region. In some embodiments, constitutive controlregions include, for example, the cytomegalovirus (CMV) control region,simian virus 40 (SV40) early control region, mouse mammary tumor virus(MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR)control region, MoMuLV control region, an avian leukemia virus controlregion, an Epstein-Barr virus immediate early control region, a Roussarcoma virus control region, as well as human control regions such as,but not limited to, the actin control region, the myosin control region,the elongation factor-1a control region, the hemoglobin control region,and the creatine kinase control region. In some embodiments, thereporter gene is under the control of an inducible control region. Insome embodiments, the inducible control regions include, for example, ametallothionein control region, a glucocorticoid control region, aprogesterone control region, a tetracycline control region, a c-foscontrol region, the T-REx system of ThermoFisher which places expressionfrom the human cytomegalovirus immediate-early control region under thecontrol of tetracycline operator(s), and the RheoSwitch control regionsof Intrexon.

In some embodiments, the target cell is modified to contain a reportergene using CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, or equivalentsystems. In some embodiments, the reporter gene is inserted at a safeharbor site within the target cell. In some embodiments, the constructcarrying the reporter gene includes a selectable marker that is alsoinserted at a safe harbor site of the target cell. In some embodiments,the selectable marker is used to select for target cells that have beenmodified with the reporter gene construct. In some embodiments, thereporter gene is inserted at the locus for a cell surface antigenrecognized by a CAR, DE-CAR, and/or Side-CAR polypeptide(s). In someembodiments, the reporter gene is inserted at both alleles for a cellsurface antigen recognized by a CAR, DE-CAR, and/or Side-CARpolypeptide(s). In some embodiments, the reporter gene inserted at thecell surface antigen allele is different from the reporter gene insertedat a different site in other target cells. In some embodiments, thetarget cells with a reporter gene inserted at the cell surface antigenallele are used as a control for the cell killing assay.

In some embodiments, the target cell is an adherent cell and thereporter gene is stably integrated into a chromosome of the target cell.In some embodiments, target cells are grown into a monolayer and thenexposed to a cell killing agent including, for example, host cells witha CAR device, Smart CAR device, DE CAR device, Smart-DE CAR device,and/or Side-CAR devices. When the cell killing agent lyses a target cellthe reporter contained therein is released into the cell media. Thereleased reporter can then be measured to determine the amount of targetcell killing. In some embodiments, the reporter is measured in the cellmedia while the cell killing is occurring. In some embodiments, themeasurements of reporter are made in real time. In some embodiments, thereporter interacts with reagents to make the detected signal. In someembodiments, the reagents are impermeable or poorly permeable to thecell membrane of the target cells. In these embodiments, the targetcells do not need to be removed from the solution to measure reporterreleased by cell killing. In some embodiments, reporter measurements areperformed on the well with the adherent target cells in a manner thatexcludes the adherent target cells from the measurement. For example, ifthe adherent layer of target cells is at the bottom of the well,reporter measurements could be taken through the well above the adherentcells. In some embodiments, these measurements are performed in realtime. In some embodiments, the reporter is an optical reporter and theoptical reading device measures the reporter in the media by reading asection of container that does not include the monolayer of targetcells.

In some embodiments, the target cell is a suspension cell with thereporter gene stably integrated into a chromosome of the target cell. Inthis embodiment, the target cells are prepared at a certain density andthen combined with a cell killing agent including, for example, hostcells containing CAR device, Smart CAR device, DE CAR device, Smart-DECAR device, and/or Side-CAR devices. When the cell killing agent lyses atarget cell the reporter contained therein is released into the cellmedia. The released reporter can then be measured to determine theamount of target cell killing. If reagents for the reporter areimpermeable or poorly permeable to the target cell membrane, thenmeasurements of the reporter released from the target cells may beperformed directly on the solution with the target cells. In someembodiments, these measurements of reporter are performed in real time.In some embodiments, the solution with the target cells is subjected toa treatment to separate the live targets from the reporter in solution.After this treatment, the reporter in the solution may be measured.

In some embodiments, the reporter is an enzyme and the reagents thatinteract with the enzyme to produce the detectable signal areimpermeable to the target cell membrane. In this embodiment, themeasurement of the reporter can occur in the presence of target cells asonly reporter that has been released by target cells will be measured.In some embodiments, the reporter reacts with another agent to bedetected and this agent is impermeable to the target cell membrane. Inthis embodiment, the measurement of reporter can occur in the presenceof target cells because the agent which detects the reporter can onlyreact with reporter that has been released by the target cells. In someembodiments, the media is separated from the target cells, andoptionally the host cells with the CAR device, Smart CAR device, DE CARdevice, Smart-DE CAR device, and/or Side-CAR devices, and then thereporter is measured in the separated media.

In some embodiments, the host cell for the CAR device, Smart CAR device,DE CAR device, Smart-DE CAR device, and/or Side-CAR devices is alsoengineered with a reporter gene. In some embodiments, the host cell isengineered with a different reporter gene than that engineered into thetarget cell. In some embodiments, the reporter engineered into the hostcells is measured and used to normalize the signal from the reporterreleased from the lysed target cells. In some embodiments, the reporterin the killing cell is used to measure the amount of killing cells addedto the target cells in the target cell killing assay. In someembodiments, the killing cell is a T-lymphocyte, a natural killer cell,or a B-lymphocyte. In some embodiments, the killing cell contains a CARdevice, Smart CAR device, DE CAR device, Smart-DE CAR device, and/orSide-CAR device. In some embodiments, the reporter in the killing cellis secreted from the killing cell. In this embodiment, the amount ofreporter secreted can correlate with the quantity of killing cells. Insome embodiments, the different reporter engineered into the host cellis detected separately from the reporter released from the target cells.In some embodiments, the target cell and control cell or a differenttarget cell have different reporters engineered into the cells. The twoor more reporters could be fluorescent and/or luminescent proteins thatproduce light at different wavelengths. Such paired light producingreporters are commercially available from ThermoFisher Scientific asdual-spectral luciferase pairs for multiplex detection. For example,among other combinations, ThermoFisher Scientific multiplexes RedFirefly luciferase (640 nm) with Renilla luciferase (460 nm), RedFirefly luciferase (640 nm) with Green Renilla luciferase (525 nm),Gaussia luciferase (470 nm) with Red Firefly luciferase (640 nm), orCypridina luciferase (470 nm) with Red Firefly luciferase (640 nm). Insome embodiments, the host cell is engineered with an enzyme reporterthat is paired with a cell membrane permeable substrate that produceslight at a different wavelength from the reporter used in the targetcells. In these embodiments, the tested sample(s) is subjected todifferent conditions that activate the reporters from the target celland/or host cell to produce signal, and those signals are measured. Insome embodiments, a fluorescent protein is used as a reporter in thehost cell and a bioluminescent protein is used as the reporter in thetarget cell. In some embodiments, the reporters are measured separately,and in some embodiments, the reporters are measured at the same time.

In some embodiments, another cell is engineered with a differentreporter gene for the cell killing assay. In some embodiments, thisother cell is a control cell, for example, if the target cell is acancerous cell, the control cell can be a normal tissue cell, or acancer cell in which the antigen target for the CAR device, Smart CARdevice, DE CAR device, Smart-DE CAR device, and/or Side-CAR devices hasbeen knocked out by a double allele knockout. In some embodiments, thetarget cell is a diseased cell, and the control cell is a normal tissuecell. In some embodiments, a cell killing agent, including, for example,host cells with CAR device, Smart CAR device, DE CAR device, Smart-DECAR device, and/or Side-CAR devices, is added to the target cells andcontrol cells and after a specific time period the reporters from thetarget cells and control cells are measured. In some embodiments, thisdual reporter data will provide a measure of the specificity of targetcell killing.

Target Cells

Target cells can be any cell type including eukaryotic cells orprokaryotic cells. In some embodiments, target cells are mammaliancells, such as, for example mouse cells, rat cells, dog cells, catcells, or human cells. In some embodiments, target cells include, forexample, cancer cells, tumor cells, hematopoietic cells, inflammatorycells, cardiovascular cells, pancreatic cells, cells from other organsin a mammal, or cells infected by a viral, bacterial, fungal, orprotozoan pathogen.

In some embodiments, the target cell is a cancer cell. In someembodiments, the cancer cell is a sarcoma, carcinoma, melanoma,chordoma, malignant histiocytoma, mesothelioma, glioblastoma,neuroblastoma, medulloblastoma, malignant meningioma, malignantschwannoma, leukemia, lymphoma, myeloma, myelodysplastic syndrome,myeloproliferative disease. In some embodiments, the cancer cellexpresses one or more of the antigens 4-1BB, 5T4, adenocarcinomaantigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125,carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200,CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40,CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR,EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factorreceptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6,insulin-like growth factor I receptor, alpha 5β1-integrin, integrinαvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid,NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostatic carcinoma cells,RANKL, RON, ROR1, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF β2,TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1,VEGFR2, 707-AP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IXantibody, CAMEL, CAP-1, CASP-8, CD25, CDC27/m, CDK4/m, CT, Cyp-B, DAM,ErbB3, ELF2M, EMMPRIN, EphA3, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE,HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IL-2R,IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R,myosin/m, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 minorbcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1or SART-3, survivin, TPI/m, TRP-1, TRP-2, TRP-2/INT2, WT1, NY-Eso-1 orNY-Eso-B or vimentin.

In some embodiments, the target cell is a cell involved in aninflammatory disease and the cell expresses one of more of the antigens,including, for example, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1),CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor),CD25 (α-chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgEFc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5,IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, Lama glama, LFA-1(CD11 a), MEDI-528, myostatin, OX-40, rhuMAb .beta.7, scleroscin, SOST,TGF β1, TNF-α or VEGF-A.

In some embodiments, the target cell is from a neuronal disorderexpressing one or more of beta amyloid or MABT5102A. In someembodiments, the target cell is involved in diabetes and expressed oneor more of L-1β or CD3. In some embodiments, the target cells is from acardiovascular disease expressing one or more of C5, cardiac myosin,CD41 (integrin α-IIb), fibrin II, beta chain, ITGB2 (CD18) andsphingosine-1-phosphate.

In some embodiments, the target cell is a Chlamydophila (Chlamydia),Ehrlichia, Rickettsia, Neisseria, Brucella, Mycobacterium, Listeria,Francisella, Legionella, Yersinia, Nocardia, Rhodococcus, Coxiella,Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, orSalmonella. In some embodiments, the target cell is a mammalian cellinfected with one of the foregoing bacteria. In some embodiments, thetarget cell is Staphylococcus aureus, Neisseria gonorrhoeae,Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus(Streptococcus agalactiae), Streptococcus pneumoniae, and Clostridiumtetani. In some embodiments, the target cell is an infectious pathogen.In some embodiments, the target cell is infected with an infectiouspathogen (e.g., a virus, a bacteria, a protozoan, or a fungus). In someembodiments, target cells are infected with bacterial pathogensincluding, for example, Helicobacter pyloris, Legionella pneumophilia, abacterial strain of Mycobacteria spp. (e.g. M. tuberculosis, M. avium,M. intracellulare, M. kansaii, or M. gordonea), Neisseria meningitides,Listeria monocytogenes, R. rickettsia, Salmonella spp., Brucella spp.,Shigella spp., or certain E. coli strains or other bacteria that haveacquired genes with invasive factors. In some embodiments, the pathogenis a eukaryote including, for example, a Histoplasma, Cryptococcus,Trypanosoma, Apicomplexans (e.g., Plasmodium), and/or Pneumocystis. Insome embodiments, target cells are infected with viral pathogensincluding, for example, Retroviridae (e.g. human immunodeficiencyviruses such as HIV-1 and HIV-LP), Picornaviridae (e.g. poliovirus,hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, andechovirus), rubella virus, coronavirus, vesicular stomatitis virus,rabies virus, ebola virus, parainfluenza virus, mumps virus, measlesvirus, respiratory syncytial virus, influenza virus, hepatitis B virus,parvovirus, Adenoviridae, Herpesviridae [e.g. type 1 and type 2 herpessimplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), andherpes virus], Poxviridae (e.g. smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus.

In some embodiments, the target cell is involved in a hematopoieticdisorder such as a malignancy or an autoimmune disease. In someembodiments, the cell is a leukemia cell, lymphoma cell, myeloma cell,or sarcoma cell. In some embodiments, the target cell is a multiplemyeloma cell. In some embodiments, the target cell is a CD19 and/or CD20positive B-cell. In some embodiments, the leukemia cells include, forexample, acute lymphoblastic leukemia cells (ALL), acute myelogenousleukemia cells (AML), chronic lymphocytic leukemia cells (CLL), chronicmyelogenous leukemia cells (CML), acute monocytic leukemia cells (AMoL),B-cell prolymphocytic leukemia cells, Hairy cell leukemia cells, T-cellprolymphocytic leukemia cells, T-cell large granular lymphocyticleukemia cells, and NK-cell leukemia cells. In some embodiments,lymphoma target cells include, for example, Hodgkin's lymphoma cells,Non-Hodgkin's lymphoma cells, lymphoplasmacytic lymphoma cells, Splenicmarginal zone lymphoma cells, small B-cell lymphoma cells, Waldenströmmacroglobulinemia cells, MALT lymphoma cells, Nodal marginal zonelymphoma cells, Pediatric follicular lymphoma cells, Mantle celllymphoma cells, Diffuse large B-cell lymphoma cells (DLBCL), largeB-cell lymphoma cells, Plasmablastic lymphoma cells, Burkitt lymphomacells, and T-cell lymphoma cells. In some embodiments, myeloma targetcells include, for example, multiple myeloma cells, and plasma cellmyeloma cells. In some embodiments, sarcoma target cells include, forexample, Histiocytic sarcoma cells, dendritic cell sarcoma cells, andLangerhans cell sarcoma cells.

In some embodiments, the target cell is from an acute myeloid leukemia(AML) expressing antigens including but not limited to any one or moreof CD 33, CD 34, CD 38, CD 44, CD 45, CD 45RA, CD 47, CD 64, CD 66, CD123, CD 133, CD 157, CLL-1, CXCR4, LeY, PR1, RHAMM (CD 168), TIM-3,and/or WT1. In some embodiments, the target cell is from a B-cellmalignancy expressing antigens including, for example, CD5, CD 10, CD19, CD 20, CD 21, CD 22, CD 23, CD 43, and CD79a. In some embodiments,target cells are from T-cell malignancies expressing antigens including,for example, CD2, CD3, CD4, CD5, CD7, and CD8. In some embodiments,target cells are from NK cell malignancies expressing antigensincluding, for example, CD 16 and CD 56. In some embodiments, targetcells are from other myeloid malignancies expressing antigens including,for example, CD13, CD33, CD 38, and CD117. In some embodiments, targetcells are from dendritic cell malignancies expressing antigensincluding, for example, CD 11c and CD123. In some embodiments, targetcells are monocyte malignancies expressing antigens including, forexample, CD 14 and CD 33.

In some embodiments, target cells are from hairy cell leukemiasexpressing antigens including, for example, CD 11, CD 19, CD 22, CD 25,and CD 103. In some embodiments, target cells are from splenic marginalzone lymphoma expressing antigens including, for example, CD19, CD22,and FMC7. In some embodiments, target cells are from lymphoplasmacyticlymphoma expressing antigens including, for example, B19, FMC7, andCD38. In some embodiments, target cells are from follicular lymphomaexpressing antigens including, for example, CD19, CD22, CD23, and CD10.In some embodiments, target cells are hematopoietic stem cellsexpressing antigens including, for example, CD 34, CD 41, CD 45, CD 90,CD 117, CD 123, and CD 133.

In some embodiments, the target cell is from an autoimmune disease, suchas, for example a neurological disorder (e.g., multiple sclerosis), arheumatological disorder (e.g., rheumatoid arthritis, systemicsclerosis, systemic lupus), a hematological immunocytopenia (pure redcell aplasia, immune thrombopenia, pure white cell aplasia), or agastrointestinal disorder (inflammatory bowel disease). In someembodiments, the neurological disorders include, for example, multiplesclerosis, myasthenia gravis, polyneuropathy, cerebellar degeneration,Guillain Barré syndrome, and amyotrophic lateral sclerosis. In someembodiments, rheumatological disorders include, for example, rheumatoidarthritis, systemic sclerosis, juvenile idiopathic arthritis, systemiclupus, erythematosus, dermatomyositis, mixed connective tissue disease,Bechet's disease, psoriatic arthritis, Ank. Spondylitis, Wegner'sgranulomatosis, and Cryoglobulinemia. In some embodiments, hematologicalimmunocytopenias include, for example, immune thrombopenia, pure redcell aplasia, autoimmune hemolytic anemia, thrombotic thrombocytopenicpurpura, Evan's syndrome, pancytopenia, and pure white cell aplasia.

In some embodiments, the target cells are memory T-cells, memoryB-cells, and hematopoietic stem cells. In some embodiments, target cellsare memory T-cells expressing antigens including, for example, CCR5,CCR7, CD11a, CD27, CD28, CD45RA, CD45RO, CD57, and/or CD62L. In someembodiments, the target cells are memory B-cells expressing antigensincluding, for example, CD 19, CD 21, CD 27, CD 40, and/or CD84. In someembodiments, the target cell is a hematopoietic stem cell expressingantigens including, for example, CD 34, CD 41, CD 45, CD 90, CD 117, CD123, and CD 133. In some embodiments, the target cell is a hematopoieticstem cell expressing antigens, including, for example, CD13, CD33, CD44, CD 47, CD 96, Mpl, Flt3, Esam1, Robo4, and/or TIM3.

In some embodiments, the target cells are senescent cells. In someembodiments, the target cells are senescent cells expressing antigensincluding, for example, DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG,LANCL1, VPS26A, or PLD3.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter. Unless otherwise indicated, thedisclosure is not limited to specific procedures, materials, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

EXAMPLES Example 1. Control of T-Cell Effector Activity with a Smart-CAR

A Smart CAR is made using the third generation anti-CD20 CAR cassettedescribed in Budde 2013 (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742, which is herebyincorporated-by-reference in its entirety for all purposes), and the RNAcontrol device, 3XL2bulge9 (Win and Smolke 2007 Proc. Natl Acad. Sci.104 (36): 14283-88, which is hereby incorporated by reference in itsentirety for all purposes). A nucleic acid encoding the 3XL2bulge9control device is engineered into the anti-CD20 CAR cassette in anappropriate expression vector.

This anti-CD20 Smart CAR is transfected by routine methods into T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD20 Smart CARs(CD20⁻/CD22⁻/CD3⁺) are activated by co-incubation with anti-CD3/CD28beads.

Activated anti-CD20 Smart CAR T-cells are co-cultured withCD20⁺/CD22⁺/CD3⁻ Ramos target cells at Smart CAR T-cell:Ramos targetratios of 2:1, 5:1, and 10:1. Ligand for the RNA control device,theophylline is added to the culture medium at concentrations in therange of 500 μM to 1 mM (lower or greater concentrations can be used totitrate Smart-CAR activity to the desired level). The Smart-CAR T-cellsand the Ramos cells are grown together for 48 hours. Cultures arewashed, and then stained with anti-CD22 and anti-CD3 reagents, followedby counting of CD22⁺ (Ramos target cells) and CD3⁺ cells (Smart CART-cells). These measurements will identify the target cell killing rate(e.g., half-life) and the proliferation rate of the Smart-CAR T-cells atdifferent levels of Smart-CAR expression.

Example 2. Control of T-Cell Effector Activity with CombinationSmart-CARs in a Human Subject

Nucleic acids encoding orthogonal Smart CARs that have specificity fordistinct TAAs and respond to distinct small molecule ligands areconstructed and are packaged into lentiviral vectors. Each of theseSmart CARs demonstrate in vitro cytotoxic T-cell effector function andantigen-dependent expansion in response to cognate ligand exposure, andindividually have known therapeutic windows in human patients.

To treat a human subject with tumors that express the defined set ofmultiple TAAs that are recognized by this Smart CAR pool, autologousT-cells are harvested from a patient's peripheral blood by apheresis andtransduced ex vivo with lentivirus encoding the cognate Smart CARs,either individually or in pools. Expanded Smart CAR CD4+ and/or CD8+T-cells are then adoptively transferred back into the patient. EachSmart CAR is individually activated with its own cognate small moleculeligand to initiate tumor recognition and elimination. As each Smart CARis individually controlled, therapeutic windows for each Smart CAR areadjusted to enforce maximal graft vs. tumor response, with tolerablegraft vs. host response. If the escape phase of tumor immunoediting isreached, the Smart CAR targeting the lost TAA is inactivated by removalof its cognate ligand to limit further graft vs. host response for aSmart CAR that no longer provides graft vs. tumor benefits. Bycontrolling Smart CAR toxicity and parallelizing a distributed attack onTAAs quickly, durable remissions for any tumor type are achieved.

Example 3. Control of T-Cell Effector Activity with a DE-CAR

A DE-CAR is made using the anti-CD20 CAR cassette described in Budde2013 (Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, whichis hereby incorporated-by-reference in its entirety for all purposes),and the destabilizing element (DE) ecDHFR described in Iwamoto 2010(Iwamoto et al. Chemistry and Biology, 2010doi:10.1016/j.chembiol.2010.07.009, which is hereby incorporated byreference in its entirety for all purposes). In an embodiment, theDE-CAR also encodes the RNA control device, 3XL2bulge9 (Win and Smolke2007 Proc. Natl Acad. Sci. 104 (36): 14283-88, which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the DE of mutant scDHFR is engineered into the anti-CD20CAR cassette in an appropriate expression vector. In an alternateembodiment, a nucleic acid encoding the 3XL2bulge9 control device isfurther engineered into the DE-anti-CD20 CAR cassette.

This anti-CD20 DE-CAR is transfected by routine methods into T-cells(Jurkat cells and/or primary human T-cells), and stable populations ofT-cells are selected using appropriate antibiotics (or other selectionschemes). T-cell populations with anti-CD20 DE-CARs or anti-CD20Smart-DE-CARs (CD20⁻/CD22⁻/CD3⁺) are activated by co-incubation withanti-CD3/CD28 beads.

Activated anti-CD20 DE-CAR T-cells or anti-CD20 Smart-DE-CAR T-cells areco-cultured with CD20⁺/CD22⁺/CD3⁻ Ramos target cells at DE-CAR T-cell(or Smart-DE-CAR T-cell):Ramos target ratios of 2:1, 5:1, and 10:1.Ligand for the DE, trimethoprim, and/or ligand for the RNA controldevice, theophylline, is added to the culture medium at concentrationsin the range of 500 μM to 1 mM (lower or greater concentrations can beused to titrate Smart-CAR activity to the desired level). The DE-CART-cells or Smart-DE-CAR T-cells and the Ramos cells are grown togetherfor 48 hours. Cultures are washed, and then stained with anti-CD22 andanti-CD3 reagents, followed by counting of CD22⁺ (Ramos target cells)and CD3⁺ cells (DE-CAR and/or Smart-DE-CAR T-cells). These measurementswill identify the target cell killing rate (e.g., half-life) and theproliferation rate of the Smart-CAR T-cells at different levels ofSmart-CAR expression.

Example 4: Control of T-Cell Effector Activity with Combination DE-CARsand/or Smart-DE-CARs in a Human Subject

Nucleic acids encoding orthogonal DE-CARs and/or Smart-DE-CARs that havespecificity for distinct TAAs and respond to distinct small moleculeligands are constructed and are packaged into lentiviral vectors. Eachof these DE-CARs and/or Smart-DE-CARs demonstrate in vitro cytotoxicT-cell effector function and antigen-dependent expansion in response tocognate ligand exposure, and individually have known therapeutic windowsin human patients.

To treat a human subject with tumors that express the defined set ofmultiple TAAs that are recognized by this DE-CAR and/or Smart-DE-CARpool, autologous T-cells are harvested from a patient's peripheral bloodby apheresis and transduced ex vivo with lentivirus encoding the cognateDE-CARs and/or Smart-DE-CARs, either individually or in pools. ExpandedDE-CAR and/or Smart-DE-CAR CD4+ and/or CD8+ T-cells are then adoptivelytransferred back into the patient. Each DE-CAR and/or Smart-DE-CAR isindividually activated with its own cognate small molecule ligand(s) toinitiate tumor recognition and elimination. As each DE-CAR and/orSmart-DE-CAR is individually controlled, therapeutic windows for eachDE-CAR and/or Smart-DE-CAR is adjusted to enforce maximal graft vs.tumor response, with tolerable graft vs. host response. If the escapephase of tumor immunoediting is reached, the DE-CAR and/or Smart-DE-CARtargeting the lost TAA is inactivated by removal of its cognate ligandto limit further graft vs. host response for a DE-CAR and/orSmart-DE-CAR that no longer provides graft vs. tumor benefits. Bycontrolling DE-CAR and/or Smart-DE-CAR toxicity and parallelizing adistributed attack on TAAs quickly, durable remissions for any tumortype are achieved.

Example 5: Integration of a Nucleic Acid Encoding a DE-CAR and/orSmart-DE-CAR at the CCR5 Locus of a Human T-Lymphocyte

The CRISPR system is used to engineer human T-lymphocyte with a nucleicacid encoding a DE-CAR and/or Smart-DE-CAR of the invention downstreamfrom an appropriate control region comprising, e.g., a promoter fromSV40, CMV, UBC, EF1A, PGK or CAGG (Qin et al (2010) PLoS ONE.doi:10.1371/journal.pone.0010611, which is incorporated by reference inits entirety for all purposes), optionally a suitable enhancer, e.g.,CMV early enhancer, and optionally other suitable regulatory sequences,e.g., woodchuck hepatitis B virus post-transcriptional regulatoryelement (WPRE; Donello, Loeb, and Hope (1998) Journal of Virology, whichis incorporated by reference in its entirety for all purposes)translation initiator at short UTR (TISU; Elfakess et al (2011) NAR 39(17): 7598-7609. doi:10.1093/nar/gkr484, which is incorporated byreference in its entirety for all purposes), A-U rich elements,beta-globin 3′ UTR and poly-A sequence, SV40 3′ UTR and poly-A sequence.This expression cassette is flanked on the 3′ and 5′ sides byappropriate CCR5 sequences for break point(s) associated with syntheticguide sequences obtained using the methodology of, for example, U.S.Pat. No. 8,697,359 (which is incorporated by reference in its entiretyfor all purposes).

Cas9 is introduced to the T-lymphocyte by electroporation of Cas9 mRNA,sgRNA, and donor nucleic acid encoding the DE-CAR and/or Smart-DE-CARwith appropriate control sequences and CCR5 flanking sequences paired tothe sgRNA(s). (See, e.g., Qin et al., Genetics 115:176594 (2015); Qin etal., Genetics 115:176594 (2015), Kim et al (2014) Genome 1012-19, Kim etal. 2014 describe the Amaza Nucleofector, an optimized electroporationsystem, all three of these references are incorporated by reference intheir entirety for all purposes.) Electroporated cells are depositedinto multiwell plates and cultured in suitable media.

Representative cells are obtained and assayed by RFLP and/or sequencingto identify T-lymphocytes with DE-CAR and/or Smart-DE-CAR constructsintegrated at the CCR5 locus.

Example 6: Identification of Antigen Binding Domains from a SubjectsAntibody Repertoire

A CAR chassis is made using the third generation anti-CD20 CAR cassettedescribed in Budde 2013 (Budde et al. PLoS1, 2013doi:10.1371/journal.pone.0082742, which is herebyincorporated-by-reference in its entirety for all purposes). This CARconstruct is engineered to remove the anti-CD20 extracellular domain,and engineered to be an acceptor for cassettes of other antigen bindingdomains.

Antigen binding domains for the CAR chassis are made from the B-cellsobtained from a subject who generated an effective immune responseagainst a disease. For example, a patient who developed immunity to aninfectious disease, such as a pandemic flu virus arising from influenzaH5N1. B-cells are obtained from the peripheral blood of the immuneindividual, and optionally, memory B-cells and/or plasma cells areobtained from the peripheral blood lymphocytes. cDNA is created from themRNA of these cells, using primers and reaction conditions as disclosed,for example, in Coronella et al, Nucl. Acids Res. 28:e85 (2000), whichis incorporated by reference in its entirety for all purpose.Alternatively, following the reverse transcription step, nucleic acidsencoding expressed antibody genes may be amplified using other primersets that are well-known in the art (e.g., those described above).Separate amplifications are performed to obtain nucleic acids encodingexpressed heavy chains and expressed light chains. These pools ofnucleic acids encoding heavy chains or light chains are combined in acombinatorial fashion to create a library of nucleic acids encodingsingle chain antibodies using, for example, the techniques and methodsdescribed in Pansri et al., BMC Biotechnol. 9:6 (2009); Peraldi-Roux,Methods Molc. Biol. 907:73-83 (2012), both of which are incorporated byreference in their entirety for all purposes.

The nucleic acids encoding the library of single chain antibodies areoperably linked to nucleic acids encoding the CAR chassis describedabove. This creates a library of CARs with different antigen bindingdomains that represent the antibodies expressed in the immuneindividuals B-cells (or plasma cells and/or memory B-cells). This CARlibrary is transfected by routine methods into T-cells (Jurkat cellsand/or primary human T-cells), and stable populations of T-cells areselected using appropriate antibiotics (or other selection schemes).

CAR T-cells are diluted to small or single numbers and placed in wellsor containers and co-cultured with virus and/or viral infected cells atappropriate ratios. The Smart-CAR T-cells and the viral targets aregrown together for 48-96 hours and, optionally, wells that show growthare diluted and the growth selection with viral targets is repeated forseveral rounds. After identification of clones that have CARs whichproliferate in response to viral target, the antigen binding domains ofthese CARs and/or the CAR constructs can be isolated. The isolatedantigen binding domains are further characterized for sequence andtarget affinity. The antigen binding domains are also used to maketherapeutic antibodies for treating the infectious disease, and the CARconstructs can be used to make therapeutic T-cells for treating patientswith the infectious disease.

Alternatively, the T-cells containing the CAR library as a whole aremixed with viral targets (virus or viral infected cells) and grown for48-96 hours. T-cells with CARs that can bind to the viral target will bestimulated to proliferate, and those that don't react with the viraltarget should not grow or will grow only a small amount. In someembodiments, multiple rounds of selective growth with viral target maybe performed to enrich and select for CAR clones that bind to the viraltarget. After several rounds of enrichment/selection, individual clonesare obtained from the mixture and characterized for binding to the viraltarget. Again, clones of antigen binding domains can be used to maketherapeutic antibodies for treating the infectious disease and the CARconstructs can be used or modified for use in treatment of the disease.

Example 7: Identification of Antigen Binding Domains Using FACs Sorting

A library of candidate antigen binding domains with CAR chassis is madeaccording to Example 6. The host T-cells are engineered to include anoptical reporter (e.g., GFP) operably linked to a NFAT control region sothat reporter is expressed when the CAR construct is activated bybinding of antigen. In this embodiment, T-cells with the CAR-antigenbinding domain library are mixed with virus and/or viral infected cellsand incubated for an appropriate amount of time (for GFP expression).T-cells with antigen binding domains that bind to virus or viralinfected cell antigens are sorted from T-cells with nonbinding antigenbinding domains using a Fluorescent Activated Cell Sorter.

In some embodiments, activation of T-cells with the CAR constructs isdone through several cycles, optionally with increasing thresholds ofGFP signal to screen for those antigen binding domains that have higheraffinity for antigen.

Example 8: Identification of Antigen Binding Domains Using a Smart-CARChassis

In this example, the isolation of nucleic acids encoding antigen bindingdomains specific for an infectious disease described in Example 6 isrepeated. These nucleic acids are operably linked to a Smart-CAR chassismade from the Smart CAR made in Example 1.

The RNA control device of the Smart CAR controls the amount ofanti-infectious disease CAR made in the T-cell, and can be used toselect for antigen binding domains with certain thresholds of bindingaffinity for the target antigen. In addition, the Smart CAR constructsthat bind to the infectious disease agent can be used during therapywith CAR T-cells to control the activity of the CAR T-cells as theyfight the infectious disease. Antigen binding domains identified andcloned from the enrichment/selection can also be used in antibodytherapies for the treatment of the disease.

Example 9. Target Cell Killing Assay for a Chimeric Antigen ReceptorTargeted to CD133 (AML)

A CAR is made using the anti-CD20 CAR cassette described in Budde 2013(Budde et al. PLoS1, 2013 doi:10.1371/journal.pone.0082742, which ishereby incorporated-by-reference in its entirety for all purposes), withthe anti-CD133 mAb 293C3-SDIE is used for the extracellular element(Rothfelder et al., 2015,https://ash.confex.com/ash/2015/webprogram/Paper81121.html, which isincorporated by reference in its entirety for all purposes) replacingthe anti-CD20 extracellular domain. In an embodiment, the anti-CD133 CARalso encodes the RNA control device, 3XL2bulge9 (Win and Smolke 2007Proc. Natl Acad. Sci. 104 (36): 14283-88, which is hereby incorporatedby reference in its entirety for all purposes), and/or the destabilizingelement (DE) ecDHFR described in Iwamoto 2010 (Iwamoto et al. Chemistryand Biology, 2010 doi:10.1016/j.chembiol.2010.07.009, which is herebyincorporated by reference in its entirety for all purposes). A nucleicacid encoding the anti-CD20 CAR cassette is engineered to replace theanti-CD20 extracellular domain with the anti-CD133 element, andoptionally the RNA control device and/or the destabilizing element arealso engineered into the cassette. The anti-CD133 CAR with or withoutthe RNA control device and/or the DE are cloned into appropriateexpression vectors.

T-lymphocytes (Jurkat cells and/or primary human T-cells), or stablepopulations of T-cells are genetically modified using CRISPR/cas9 tomake a double-allele knockout of FasL. Multiple guide RNAs specific forFasL are designed and then together with the cas9 enzyme are introducedinto the T-cells. Double allele FasL knockouts are identified byT-lymphocyte clones that do not stain with anti-FasL antibody.

This anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR, and/or theanti-CD133 DE-Smart CAR are transfected by routine methods into thedouble-allele knockout FasL T-cells. T-cell populations withanti-anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR, and/or theanti-CD133 DE-Smart CAR (CD20⁻/CD22⁻/CD3⁺) are activated byco-incubation with anti-CD3/CD28 beads.

CD133⁺/CD3⁻ AML target cells (e.g., U937, MV4-11, MOLM-14, HL-60 and/orKG1a) are genetically modified with the Renilla luciferase gene underthe control of the CMV control region (pGL4.75 vector from Promega). TheRluc gene with CMV control region from pGL4.75 is engineered forinsertion by CRISPR into genome of the AML target cells (e.g., U937,MV4-11, MOLM-14, HL-60 and/or KG1a). AML target cells that are Rluc+ areidentified and used as target cells.

Activated anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR,and/or the anti-CD133 DE-Smart CAR T-cells are co-cultured with theRluc+, CD133⁺/CD3⁻ AML target cells (e.g., U937, MV4-11, MOLM-14, HL-60and/or KG1a) at anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR,and/or the anti-CD133 DE-Smart CAR T-cell:AML target ratios of 2:1, 5:1,and 10:1. Ligand for the DE, trimethoprim, and/or ligand for the RNAcontrol device, theophylline, is added to the culture medium atconcentrations in the range of 500 μM to 1 mM (lower or greaterconcentrations can be used to titrate Smart-CAR activity to the desiredlevel). The anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR,and/or the anti-CD133 DE-Smart CAR T-cells and the AML cells are growntogether for 48 hours. Aliquots of the culture are washed, and thenstained with anti-CD3 reagents, followed by counting of CD3⁺ cells(anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR, and/or theanti-CD133 DE-Smart CAR T-cells). Separate aliquots are placed incentrifuge tubes, spun to pellet the cells, and the supernatant is thentested for Rluc activity by adding appropriate reagents for the Rluc.Rluc activity is measured using an appropriate spectrophotometer. Thesemeasurements will identify the target cell killing rate (e.g.,half-life) and the proliferation rate of the anti-CD133 CAR, anti-CD133DE-CAR, anti-CD133 Smart CAR, and/or the anti-CD133 DE-Smart CAR T-cellsat different levels of CAR and/or DE-CAR expression.

Example 10. Target Cell Killing Assay with Labeled Target Cells andLabeled Host Cells

T-lymphocytes (Jurkat cells and/or primary human T-cells), or stablepopulations of T-cells are genetically modified using CRISPR/cas9 tomake a double-allele knockout of FasL, and a double allele insertion ata safe harbor site of a nucleic acid encoding GFP under the control of asuitable promoter (e.g., CMV). Multiple guide RNAs specific for FasL andthe safe harbor site/GFP construct are designed and then together withthe cas9 enzyme are introduced into the T-cells. Double allele FasLknockouts are identified by T-lymphocyte clones that do not stain withanti-FasL antibody.

These FasL−, GFP+ T-lymphocytes are used as host cells for theanti-CD133 CAR described in Example 2. FasL−, GFP+ T-lymphocytepopulations with anti-anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133Smart CAR, and/or the anti-CD133 DE-Smart CAR (CD20⁻/CD22⁻/CD3⁺) areactivated by co-incubation with anti-CD3/CD28 beads.

Activated anti-CD133 CAR, anti-CD133 DE-CAR, anti-CD133 Smart CAR,and/or the anti-CD133 DE-Smart CAR T-cells are co-cultured with theRluc+, CD133⁺/CD3⁻ AML target cells of Example 2, under similarconditions to those of Example 2. After the appropriate incubation time,the host cells are measured by GFP fluorescence after excitation of GFPwith 360-400 nm spectrum of light that excites GFP fluorescence. Targetcell killing is measured by adding the appropriate reagents for Rluc andmeasuring the bioluminescence from the Rluc released from killed targetcells.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for finding a CAR that is activated byan antigen, comprising the steps of: providing a plurality of eukaryoticcells wherein each eukaryotic cell comprises a nucleic acid wherein thenucleic acid comprises a polynucleotide encoding an antigen bindingdomain; a polynucleotide encoding a CAR chassis, wherein the CAR chassisis comprised of a transmembrane element and an intracellular element,wherein the transmembrane element is linked to the intracellularelement, and wherein the polynucleotide encoding the antigen bindingdomain is operably linked to the polynucleotide encoding the CARchassis, and wherein the plurality of eukaryotic cells collectivelydisplay a plurality of different antigen binding domains; mixing theantigen with the plurality of eukaryotic cells; incubating the pluralityof eukaryotic cells with the antigen; and identifying a eukaryotic cellthat is activated in the presence of the antigen, whereby the CAR thatis activated by the antigen is identified.
 2. The method of claim 1,further comprising the steps of: taking an aliquot of eukaryotic cellsfrom the first incubation, mixing the aliquot of cells with the antigen,and incubating the eukaryotic cells with the antigen.
 3. The method ofclaim 2, further comprising the steps of: taking an aliquot ofeukaryotic cells from the second incubation, mixing the aliquot of cellswith the antigen, and incubating the eukaryotic cells with the antigen.4. The method of claim 1 wherein the polynucleotides encoding theantigen binding domains are derived from a plurality of nucleic acidsobtained from a subject wherein the subject had an immune response tothe antigen.
 5. The method of claim 1, wherein the antigen is part of aninfectious disease causing agent, and wherein the antigen mixed with theeukaryotic cell includes the infectious disease causing agent.
 6. Themethod of claim 5, wherein the antigen is not known.
 7. The method ofclaim 1, wherein the antigen is part of a cancer cell, and wherein theantigen mixed with the eukaryotic cell includes the cancer cell.
 8. Themethod of claim 7, wherein the antigen is not known.
 9. The method ofclaim 1, wherein the eukaryotic cell is a mammalian cell.
 10. The methodof claim 1, wherein the eukaryotic cell is a T-lymphocyte.
 11. Themethod of claim 1, wherein the eukaryotic cell is a natural killer cell.12. The method of claim 1, wherein the eukaryotic cell is aB-lymphocyte.
 13. The method of claim 1, further comprising the stepsof: obtaining a clone of the eukaryotic cell and the CAR that areactivated by the antigen.
 14. The method of claim 1, wherein the nucleicacid of claim 1 further comprises a polynucleotide encoding an RNAcontrol device and wherein the polynucleotide encoding the RNA controldevice is operably linked to the polynucleotide encoding the CARchassis.
 15. The method of claim 1, wherein the nucleic acid of claim 1further comprises a polynucleotide encoding a destabilizing element andwherein the polynucleotide encoding the destabilizing element isoperably linked to the polynucleotide encoding the CAR chassis.
 16. Themethod of claim 1, wherein the antigen binding domain is derived from anantibody.
 17. The method of claim 16, wherein the antibody is a singlechain antibody.
 18. The method of claim 1, wherein the antigen bindingdomain is derived from a T-cell receptor.
 19. The method of claim 18,wherein the T-cell receptor is a single chain T-cell receptor.
 20. Themethod of claim 1, wherein the antigen binding domain is an innateimmunity receptor.